Twenty-two HIV-1-infected Russian individuals were studied to gain better insight on the genetic and biological characteristics of HIV-1 variants present in Russia. The V3 domain of the HIV-1 envelope was directly sequenced from cultured peripheral blood mononuclear cells (PBMCs). Phylogenetic analyses were used to determine the HIV-1 genetic subtype and to study transmission patterns. Virus isolates were obtained from PBMCs and the biological phenotype was determined by coculture with MT-2, CEM, and Jurkat-tat cells. Twelve homo- and bisexual men carried subtype B variants, whereas 6 heterosexually infected individuals carried subtype F virus. A heterosexual man infected in the Ivory Coast and a nosocomially infected young female carried subtype A and G virus, respectively. Interestingly, phylogenetic analyses suggested that subtype B may have entered Russia on at least four occasions and, even more surprisingly, that the relatively rare subtype F has entered Russia on at least three occasions. Two suspected transmission clusters of subtype F were supported by the phylogenetic analyses, whereas one suspected subtype B transmission cluster was not. Positively charged amino acids in positions 311 and 325 of V3 loop have been shown to be important determinants for the MT-2-positive biological phenotype of virus isolates. Interestingly, we found that the direct PBMC sequences from individuals with MT-2-positive isolates displayed specific neutral, instead of positively charged, amino acids, in these positions. This indicates that it may be more difficult to predict the biological phenotype of HIV-1 using sequences from uncultured PBMCs than from virus isolates.