Scottish schools must provide separate toilets for boys and girls on the basis of biological sex, the government has said.
However, new guidance issued by Holyrood ministers says schools can also provide gender neutral toilets for transgender students.
Schools had previously been told that pupils could use whichever toilet they felt most comfortable in.
The updated guidance follows two landmark court rulings relating to single-sex spaces.
In April, a judge ruled that schools in Scotland must provide single-sex toilets to pupils after some provided only gender neutral facilities.
That came a week after the UK’s Supreme Court unanimously ruled that the definition of a “woman” and “sex” in the Equality Act 2010 refers to “a biological woman and biological sex”.
Monday’s new guidance for schools says that “separate toilet facilities for boys and girls must be provided in schools”.
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It adds: “As the law stands, the facilities require to be made available on the basis of biological sex.”
It says schools should “consider” facilities for transgender pupils which “may include the use of gender neutral provision”.
The guidance additionally warns against the risk of “outing” a young person as transgender and urges schools to support those who “want to live as a boy although their biological sex was female, or they now want to live as a girl, although their biological sex was male”.
It says that denying this would have a “detrimental impact on the young person’s wellbeing, relationships and behaviour”.
It adds: “This may mean that it is necessary that practical arrangements such as enabling young people to use facilities outwith usual breaktimes, or for particular facilities to be available aligned to the young person’s activities within school, to reduce visibility of them moving across and within the school building to access toilet or changing room facilities.
“Depending on the particular circumstances of individual pupils, schools may require to take legal advice on any approach or proposed approach.”
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For school uniforms, the Scottish government also says that “forcing transgender young people to wear clothes which do not match their gender identity can be distressing for them and may constitute discrimination under the Equality Act 2010”.
The Scottish Tories said the new guidance showed the Scottish government was “dragging their heels” on new rules for single-sex spaces.
Roz McCall, the party’s spokeswoman for children and young people, said: “The Supreme Court ruling was clear, the legal definition of a woman is based on biological sex, and they are entitled to single-sex spaces.
“But instead of enforcing the law, the SNP have confused matters further by producing this contradictory and potentially harmful guidance that will make things more difficult for schools.”
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Education secretary Jenny Gilruth said the rights of all children and young people “must be respected in our schools”.
She said: “The Scottish government has made clear it accepts the Supreme Court ruling and since April has been taking forward the detailed work that is necessary as a consequence of the ruling. That work is ongoing.
“We have brought forward updates to guidance to provide clarity and confidence to teachers and staff as they work to support the mental, physical and emotional health of transgender young people in our schools following recent significant legal and policy developments.”
The guidance is not mandatory but advisory as the local authorities run the schools, not the Scottish government directly.
It has been updated in response to the legal requirements.
Ms Gilruth added: “The Scottish government respects the rights of everybody. I want every pupil to be able to reach their potential and every pupil deserves our support to do that. Our guidance means that all of their individual needs will be respected.
“All schools are required to provide separate toilets for girls and boys.
“In addition, the guidance makes clear that councils should give careful consideration to the individual needs of transgender pupils in light of the school context and school community.”
