HIV-1-positive individuals were recruited from January 1993 to December 1996 from several cohorts receiving follow-up in the city of Rio de Janeiro, Brazil, to evaluate HIV-1 genetic variability and the potential association with modes of transmission. HIV-1 subtyping was carried out using the heteroduplex mobility assay (HMA), and those samples corresponding to the typical Brazilian subtype B variant were further identified based on the Fok I restriction fragment length polymorphism (RFLP). DNA sequencing was performed to evaluate one case of subtype D infection. From the 131 HIV-1-positive individuals analyzed, 106 (80.9%) could be identified as infected by subtype B and 20 (15.3%) by subtype F. One of the samples (0.8%) was classified as subtype D. DNA samples from 4 patients (3.0%) did not yield polymerase chain reaction (PCR)-amplified products to be typed. Based on the Fok I RFLP, 39 of the 106 subtype B samples (37%) were identified as corresponding to the typical Brazilian subtype B variant containing the GWGR motif at the tip of the V3 loop. No statistically significant association could be detected between HIV-I subtypes and modes of transmission, exposure categories, or gender. This is the first reported case of HIV-1 subtype D infection in Brazil.