The effects of 3'-azido-3'-deoxythymidine (AZT) on the deoxynucleotide pools of three human cell lines, HL-60, H-9, and K-562, were determined. The corresponding ED50s for inhibition of cell growth were 670, 100, and 100 microM AZT. In all three lines, exposure to 200 microM AZT caused dTTP and dGTP initially to fall and then to return towards control levels. In contrast to a previous report [Furman et al., (1986) Proc. Nat. Acad. Sci. USA 83, 8333-8337], dCTP levels increased. Pools of dATP were relatively unchanged. Qualitatively similar changes occurred in 10 microM AZT, but recovery was faster than at 200 microM AZT. After 24 hrs incubation with 200 microM AZT, AZT-5'-MP reached 2.8, 4.7, and 15.7 mM in the HL-60, H-9, and K-562 cells, respectively. When HL-60 and K-562 cells incubated in AZT were resuspended in fresh medium, AZT-5'-MP pools declined with respective t1/2 values equal to 34 and 68 min. The concentration of thymidine, and to a lesser extent deoxyuridine, increased in the media of treated cells. AZT-5'-MP was found in the media of cells treated with AZT.