We compared the relative susceptibility of HL-60, a human acute progranulocytic leukemia cell line, and the normal human myeloid progenitor cell, the colony-forming unit in culture, to growth inhibition by thymidine. Normal human myeloid colony formation was more sensitive to thymidine than was HL-60 colony formation, the former being inhibited by greater than or equal to 0.5 mM thymidine and the latter by greater than or equal to 5.0 mM. Colony inhibition by thymidine was irreversible in both cases after a seven-day incubation of the colony-forming unit in culture in liquid medium enriched with thymidine and subsequent replating in thymidine-free soft agar. Thymidine-induced inhibition of HL-60 cloning could be partially prevented by deoxycytidine, whereas normal myeloid cloning could not, suggesting that high-concentration thymidine may affect processes necessary for cloning in addition to deoxyribonucleotide synthesis. HL-60 growth in liquid suspension culture could be suppressed transiently by 1.0 mM thymidine, suppressed totally by greater than or equal to 5.0 mM thymidine, and rescued completely by concomitant addition of deoxycytidine. The development of resistance to 1.0 mM thymidine could not be accounted for by reduction in thymidine pool size or by reduction in thymidine kinase activity. Replating of HL-60 cells growing in the presence of 1.0 mM thymidine in liquid medium in thymidine-free soft agar produced significant colony inhibition, also suggesting that thymidine affects more than just deoxyribonucleotide synthesis or that the clonogenic HL-60 cell presents a distinct subpopulation with more sensitive deoxyribonucleotide-synthetic control mechanisms.