Human immunodeficiency virus (HIV)-1 strains have been divided into three groups: main (M), outlier (O), and non-M non-O (N). Biochemical analyses of HIV-1 reverse transcriptase (RT) have been performed predominantly with enzymes derived from HIV-1 group M:subtype B laboratory strains. This study was designed to optimize the expression and to characterize the enzymatic properties of HIV-1 group O RTs as well as chimeric RTs composed of group M and O p66 and p51 subunits. The DNA-dependent DNA polymerase activity on a short heteropolymeric template-primer was similar with all enzymes, i.e. the HIV-1 group O and M and chimeric RTs. Our data revealed that the 51-kDa subunit in the chimeric heterodimer p66(M:B)/p51(O) confers increased heterodimer stability and partial resistance to non-nucleoside RT inhibitors. Chimeric RTs (p66(M:B)/p51(O) and p66(O)/p51(M:B)) were unable to initiate reverse transcription from tRNA(3)(Lys) using HIV-1 group O or group M:subtype B RNA templates. In contrast, HIV-1 group O and M RTs supported (-)-strand DNA synthesis from tRNA(3)(Lys) hybridized to any of their corresponding HIV-1 RNA templates. HIV-2 RT could not initiate reverse transcription on tRNA(3)(Lys)-primed HIV-1 genomic RNA. These findings suggest that the initiation event is conserved between HIV-1 groups, but not HIV types.