Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3'-azido-3'-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [(32)P]ddAMP-terminated DNA primer/template gave rise to (32)P-labeled adenosine 2',3'-dideoxyadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)ddA), ddATP, Gp(4)ddA, and Ap(3)ddA, corresponding to the transfer of [(32)P]ddAMP to ATP, PP(i), GTP, and ADP, respectively. Incubation with [(32)P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous (32)P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PP(i) levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4(+) or CD8(+) T cells, while PP(i) was present at 7 to 15 microM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4(+) or CD8(+) T cells contained 1.4 to 2.7 mM ATP and 55 to 79 microM PP(i). These cellular PP(i) concentrations are lower than previously reported; nonetheless, the PP(i)-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PP(i)-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.