We have expressed purified recombinant reverse transcriptase (RT) from clinical isolates of human immunodeficiency virus subtypes B, C, and A/E in Escherichia coli. The drug sensitivities of these RTs were then determined for both nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) in cell-free RT assays. Although A/E and C viruses contained numerous polymorphisms relative to subtype B (i.e., naturally occurring variations unrelated to drug resistance), the wild-type enzymes prepared from these or subtype A/E clinical isolates displayed <2-fold differences in drug sensitivities with regard to the active triphosphate active forms of NRTIs, as compared with RT expressed from BH-10 recombinant virus. Recombinant RTs from clinical isolates of subtypes B, C, and A/E that contained multiple resistance-associated mutations displayed expected variations in levels of resistance to the intracellular active forms of 3TC, ddI, ddC, and PMPA, that is, 3TCTP, ddATP, ddCTP, and PMPApp, respectively. Subtype A/E and C RT enzymes contained only minor NNRTI polymorphisms that distinguished them from wild-type subtype B enzymes and wild-type RTs from these various subtypes showed only 1- to 4-fold variability in IC(50) values for each of nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), and calanolide A. In contrast, RT enzymes from subtype B and C viruses harboring specific NNRTI mutations were highly resistant to all four tested NNRTIs. Subtype C variants containing the novel V106M resistance codon showed cross-resistance to all approved NNRTIs in cell-free RT assays.