An open cohort of 2670 homosexual men repeatedly tested for HIV at a Madrid clinic has registered 8050 person-years (PY) of follow-up and 157 seroconversions from 1988 to 2000. After declining from 1988 (4.71 per 100 PY), the incidence rate began a significant upward trend, reaching a figure of 2.16 per 100 PY in 2000. These findings ought to alert surveillance systems and prevention programmes.