The brothers' work with Iranian drug users was subject of a BBC film
A human rights group is calling on Iran to release immediately or charge two doctors renowned for their work on the prevention and treatment of HIV/Aids.
Human Rights Watch says the authorities have not disclosed why Arash Alaei and Kamyar Alaei were detained last month, or where they are being held.
The two brothers have travelled widely outside Iran, including to the US, to take part in conferences on HIV/Aids.
They were due to take part in a major meeting in Mexico next month.
Arash Alaei was scheduled to give a presentation on some of Iran's innovative HIV programmes, Human Rights Watch says.
The brothers are credited with getting the Iranian authorities to tackle the stigma of HIV infection and the disease Aids, in a country where sex, drug abuse and the disease itself are taboo subjects.
Damage to reputation
Joe Amon, Human Rights Watch's HIV/Aids programme director says Iran's work to combat the disease is internationally acclaimed, with the Alaei brothers playing an important role.
The brothers spearheaded educational campaigns among drug users and prostitutes and introduced harm reduction programmes in prisons, working closely with Iran's government and religious leaders.
Human Rights Watch says the brothers have not had legal representation and their detention was damaging Iran's reputation for tackling the Aids epidemic.
Kamyar Alaei is registered as a post-graduate at the University of Albany in the US.
In recent years Iran has arrested a number of academics who have ties with the US, its main global enemy.