Data from 23 European countries show that the annual number of HIV diagnoses in men who have sex with men (MSM) increased by 86% between 2000 and 2006. This paper reports the main preliminary results of a bio-behavioural survey in MSM with a specific focus on HIV prevalence and use of United Nations General Assembly Special Session (UNGASS) indicators in six cities in Southern and Eastern Europe. Time-location sampling (TLS) was used. A total number of 2,356 questionnaires and 2,241 oral fluid samples were collected (invalid samples 4.1%). The data show different socio-demographic patterns across countries regarding age, level of education, living conditions, living area and self-identity. Southern European cities had the highest percentage of people who had tested for HIV and collected the result. More than 50% of respondents in the sample from Barcelona reported having used a condom last time they had anal sex (57.2%), whilst in all other cities this proportion was below 50%. The cities with the highest HIV prevalence in MSM were Barcelona (17.0%) and Verona (11.8%) whilst lower percentages were reported in Bratislava (6.1%), Bucharest (4.6%), Ljubljana (5.1%) and Prague (2.6%). The low prevalence in Eastern European cities is encouraging. However, with the level of high-risk sexual behaviour documented and the lower frequency of HIV test seeking behaviour, there is a clear risk of an increase in HIV transmission.