The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is the target enzyme for the tetrahydro-imidazo[4,5,1-jk][1,4]- benzodiazepin-2(1H)one and thione (TIBO) derivatives, a class of highly potent and selective anti-HIV agents that specifically inhibit HIV-1 but not HIV-2 replication. The amino acid sequence divergence may be held responsible for the differential sensitivity of HIV-1 RT and HIV-2 RT to the TIBO derivatives. Using site-directed mutagenesis, we have introduced several amino acid substitutions in the conserved regions of HIV-1 RT. Where applicable, the amino acids were replaced by the corresponding amino acids present in HIV-2 RT. The amino acid residues Y181 and Y188 appeared to be critical for the anti-HIV-1 RT activity of the TIBO derivatives, since substitution of these residues by the corresponding HIV-2 amino acids I181 and L188 resulted in a virtual loss of TIBO sensitivity without loss of enzymatic activity.