Eight HIV-2-infected Caucasian men living in the same geographical area in Gipuzkoa (northern Spain) have been identified in the last 5 years. HIV-2 infection in this area is uncommon, and no other cases of HIV-2 infection have been found after extensive testing for HIV-1/2 antibodies. Epidemiological data suggested a possible link among the identified subjects, with homosexual contact being the most likely way of transmission. A genetic analysis of four of the subjects, from whom specimens were available, was conducted. Phylogenetic and signature pattern studies of the reverse transcriptase (RT) and env genes supported a single source of infection. Interindividual nucleotide variability ranged from 2.4 to 4.8% in the RT region and from 5.2 to 6.1% in the env gene, whereas the mean divergence between patient and control strains was 9.8 and 18.3%, respectively. The nucleotide and amino acid signature patterns were closely related in viruses from the four examined individuals. This is the first report of a cluster of HIV-2 infections with genetic sequence data support. The singularity of this cluster should alert clinicians on the possibility of HIV-2 outside endemic areas.