There is a lack of correlation between biochemical studies and the observed clinical resistance of AIDS patients on long-term AZT therapy. Mutant HIV-1 reverse transcriptase in the viral isolates from these patients shows a 100-fold decrease in sensitivity to AZT whereas little or no difference is observed in kinetic parameters in vitro using steady-state kinetic analysis. A pre-steady-state kinetic analysis was used to examine the binding and incorporation of 2'-deoxythymidine 5'-triphosphate (dTTP) and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) by wild-type HIV-1 reverse transcriptase and a clinically important AZT-resistant mutant form of the enzyme (D67N, K70R, T215Y, K219Q) utilizing a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. It was determined that with this RNA/RNA substrate there is a 2.6-fold increase in the selection for incorporation of the natural nucleotide dTTP over the unnatural nucleoside analogue AZTTP by AZT-resistant reverse transcriptase as compared to its wild-type form. This observation indicates that the tRNALys initiation step plays an important role in the development of drug resistance. Furthermore, this result implies that the structural basis of AZT resistance in HIV-1 reverse transcriptase involves the conformation of the RNA-DNA junction (formed upon attachment of a deoxynucleotide to the RNA primer). Taken together, these observations suggest a new pharmacological basis for the development of more effective and novel AIDS drugs.