S-phase-active cytotoxic drugs selectively damage leukemic cells, but the mechanisms of this action are not clear. We have investigated the previously reported potentiation of toxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) to HL-60 cells by the differentiation-inducing steroid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and compared the results with the effects of other drugs which inhibit DNA synthesis. Determination of the intracellular content of the active metabolite of ara-C, ara-CTP, excluded more prolonged retention of the drug as the basis for potentiation of cytotoxicity. Alkaline elution of replicating DNA showed that 1,25(OH)2D3 added with or immediately after ara-C or hydroxyurea reduced the rate maturation of the replicating DNA and resulted in an increased proportion of smaller DNA fragments. However, pretreatment of the cells with 1,25(OH)2D3 inhibited this effect of the drugs on replication of DNA. No direct effect of 1,25(OH)2De on replicating DNA could be detected. The results suggest that the early events which initiate cell differentiation may protect an intact DNA replicative machinery from S-phase-active drugs but reduce the rate of DNA maturation once DNA integrity has been compromised by inhibitors of DNA synthesis.