OBJECTIVE The object of this study was to investigate the antineoplastic action of 5-aza-2'-deoxycytidine (5-AZA) in combination with vitamin D analogs on HL-60 and NB-4 myeloid leukemic cells. The vitamin D analogs chosen for this investigation were 1,25-(OH)2-23-yne-cholecalciferol (23-D) and 1,25-(OH)2-delta16-23-yne-cholecalciferol (16-23-D) since they have the potential to be used clinically owing to their minimal action on calcium metabolism. METHODS HL-60 and NB-4 leukemic cells were incubated with different concentrations of 5-AZA and either 23-D or 16-23-D and their antineoplastic action determined by inhibition of DNA synthesis and growth, induction of differentiation and colony assay. RESULTS 5-AZA in combination with either vitamin D analog produced a greater growth inhibition and induction of differentiation than either agent alone. For HL-60 leukemic cells the combination of 5-AZA with either analog produced a synergistic loss of clonogenicity. These effects on clonogenicity correlated with the effects of the combination on inhibition of growth and DNA synthesis. CONCLUSIONS These results suggest that vitamin D analogs may enhance the antileukemic action of 5-AZA and that it may be interesting to test these agents in combination in patients with myeloid leukemia.