The multiple mutations associated with high-level AZT resistance (D67N, K70R, T215F, K219Q) arise in two separate subdomains of the viral reverse transcriptase (RT), suggesting that these mutations may contribute differently to overall resistance. We compared wild-type RT with the D67N/K70R/T215F/K219Q, D67N/K70R, and T215F/K219Q mutant enzymes. The D67N/K70R/T215F/K219Q mutant showed increased DNA polymerase processivity; this resulted from decreased template/primer dissociation from RT, and was due to the T215F/K219Q mutations. The D67N/K70R/T215F/K219Q mutant was less sensitive to AZTTP (IC50 approximately 300 nM) than wt RT (IC50 approximately 100 nM) in the presence of 0.5 mM pyrophosphate. This change in pyrophosphate-mediated sensitivity of the mutant enzyme was selective for AZTTP, since similar Km values for TTP and inhibition by ddCTP and ddGTP were noted with wt and mutant RT in the absence or in the presence of pyrophosphate. The D67N/K70R/T215F/K219Q mutant showed an increased rate of pyrophosphorolysis (the reverse reaction of DNA synthesis) of chain-terminated DNA; this enhanced pyrophosphorolysis was due to the D67N/K70R mutations. However, the processivity of pyrophosphorolysis was similar for the wild-type and mutant enzymes. We propose that HIV-1 resistance to AZT results from the selectively decreased binding of AZTTP and the increased pyrophosphorolytic cleavage of chain-terminated viral DNA by the mutant RT at physiological pyrophosphate levels, resulting in a net decrease in chain termination. The increased processivity of viral DNA synthesis may be important to enable facile HIV replication in the presence of AZT, by compensating for the increased reverse reaction rate.