To characterize the viruses responsible for the HIV-1 epidemic in Spain, we genetically characterized 79 samples obtained from Spanish patients with different risk practices (injecting drug users and male homosexuals) in two regions (Madrid and Navarra). Genetic characterization was carried out by nucleotide sequencing in the C2-V3-C3 region and by phylogenetic analysis. All samples were of subtype B except one that clustered with clade F. Because no segregation of samples was determined according to the risk group of patients nor to their geographic origin, the Spanish samples analyzed constitute a single group of viruses. These data, along with the starlike topology of the phylogenetic tree, support the existence of a single introduction of HIV-1 subtype B in Spain. The mean genetic distance among subtype B sequences was of 13.9%+/-0.06% (range, 5%-25%), suggesting an epidemic of long evolution. Analysis of sequences in relation to isolation dates revealed an increase in the heterogeneity of the nucleotide sequences with time. According to these data, a divergence rate of 0.49%+/-0.11% per year was calculated for the Spanish samples during the period analyzed.