We investigated the mechanism of cell growth inhibition caused by the deoxyribonucleosides thymidine (dThd), deoxyguanosine (dGuo), deoxyadenosine (dAdo), and deoxycytidine (dCyd). Growth of the cultured human leukemic cells HL-60 and K-562 was measured by cloning in soft agar. Of the deoxyribonucleosides, dGuo was the most potent cell growth inhibitor; however, the potency of added dAdo was probably attenuated by the presence of adenosine deaminase in the tissue culture growth medium. The concentrations of nucleoside causing 50% inhibition of HL-60 cloning were: dCyd, greater than 10,000 microM; dAdo, 500 microM; dThd, 5,000 microM; and dGuo, 80 microM. For K-562 cloning, the concentrations causing 50% inhibition of cloning were dCyd, greater 10,000 microM; dAdo, 1,600 microM; dThd, 880 microM;' and dGuo, 100 microM. Measurement of deoxycytidine 5'-triphosphate (dCTP) pool size in HL-60 cells following incubation with 750 microM deoxyribonucleosides revealed that dGuo caused the greatest reduction of dCTP pools, both in early (passage 10)- and late (passage 71)-passage-derived HL-60 cell cultures (35 and 19% of control, respectively), compared to dThd (61 and 26% of control, respectively) and dAdo (39% of control of HL-60 passage 10). In K-562 cells, reductions in dCTP pool size caused by dAdo, dThd, and dGuo were 68, 46, and 35% of control, respectively. Incorporation of [3H]dCyd into DNA of HL-60 and K-562 cells was enhanced by dThd and dGuo, but the degree of enhancement was greater for dThd than for dGuo. Despite its effect in reducing HL-60 dCTP pool size, dAdo failed to enhance [3H]dCyd incorporation in either HL-60 or K-562 cells. Addition of dCyd to the cultures could only partially rescue the inhibition of HL-60 cloning caused by dThd or dGuo, suggesting that inhibition of cytidine 5'-diphosphate reduction by ribonucleotide reductase is not the only mechanism whereby these nucleosides inhibit leukemic cell cloning. These data suggest that, in addition to inhibiting de novo dCTP production via ribonucleotide reductase, these nucleosides may affect other processes in the salvage pathway such as cellular uptake and phosphorylation or the DNA polymerase reaction itself.