Objective: To determine the distribution of HIV-1 subtypes in Sao Paulo, Brazil. Methods: Samples were obtained from 80 consecutive HIV-1-infected individuals attending the Immunodeficiency Clinic at the University of Sao Paulo in 1993. Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque gradient and a portion was used for routine CD4 counts; the remainder were frozen. PBMC were proteinase-K-digested and DNA-purified by organic extraction. Samples were amplified for the env region of HIV, and envelope sequence subtypes determined by heteroduplex mobility analysis using prototypic subtypes as references. A subset of these were also sequenced through the C2-V3 region of env. Results: A total 69 of 80 samples yielded env polymerase chain reaction product enabling subtype determination; samples that did not amplify were those with low DNA yields. Among 12 injecting drug users (IDU) or sexual partners of IDU, four were typed as clade F and eight as clade B. Forty-three homosexual men or female sexual partners of bisexual men were typed as clade B. The 14 additional cases without known risk factors were typed as clade B. Conclusion: These data suggest that subtype F is related to injecting drug use in Brazil.