Little is known concerning the mechanism of myeloid differentiation. A human promyelocytic cell line (HL-60) differentiates to granulocytes or macrophage-like cells when cultured with a variety of agents. How these agents trigger myeloid differentiation is not understood. This study shows that 1.0-10.0 micrograms/ml bromodeoxyuridine (BrdU) induced myeloid differentiation of HL-60 in liquid culture. After 7 days, BrdU (3.0 micrograms/ml) produced only moderate inhibition of HL-60 growth, but induced myeloid maturation with 40% of the cells becoming morphologically more mature; 41% developed the ability to reduce nitroblue tetrazolium (NBT); 19% phagocytized Candida albicans; and 18% developed Fc receptors. The action of BrdU was mimicked by 5-iodo-deoxyuridine. Thymidine (Td) (1- to 10-fold excess) competitively inhibited incorporation of [3H]BrdU into DNA of HL-60 and inhibited the triggering of HL-60 differentiation by BrdU. The BrdU-induced maturation of HL-60 correlated with the incorporation of BrdU into DNA of HL-60. DNA buoyant density studies showed that about 46% of the Td was replaced by BrdU in each DNA strand of HL-60 as the cells differentiated in culture containing 3 micrograms/ml BrdU for 7 days. We established 20 thymidine kinase (TK)-deficient HL-60 clones. The HL-60 TK-deficient cells were unable to phosphorylate Td, to incorporate either [3H]Td or [3H]BrdU or differentiate in the presence of BrdU (1-1000 micrograms/ml). The HL-60 TK-deficient cells retained the ability to differentiate in the presence of other HL-60 inducers. Taken together, the studies suggest myeloid differentiation of HL-60 is triggered because of incorporation of BrdU into DNA of the cells.