A retrospective cohort study of 2124 repeat HIV testers attending at anonymous testing sites (ATS) of Catalonia between 1995 and 2001 registered 3273 person-years (PY) of follow-up and 76 seroconversions (incidence density of 2.32 per 100 PY; 95% CI: 1.83, 2.90). The highest HIV infection incidence was observed among heterosexual injection drug users (IDU) (9.25 per 100 PY; 95% CI: 6.77, 12.25) and the lowest was among heterosexual non-IDU (0.69 per 100 PY; 95% CI: 0.35, 1.24). In multivariate Cox regression, factors independently associated with seroconversion were men having sex with men, IDU, and having sex with a known HIV-infected person. In spite of some limitations, such results suggest that prevention interventions targeting specific groups attending at ATS in Catalonia should be prioritized.