One of the most readily quantitated indices of myeloid maturation in HL-60 cells is their ability to respond to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity. HL-60 cells exposed to the antileukemic drug, 5-azacytidine (3 to 5 µm) for 24 hr and subsequently cultured in its absence for 2 to 3 days develop an enhanced ability to respond to 12-O-tetradecanoylphorbol-13-acetate with increased respiratory burst activity detectable as an increase both in hexose monophosphate shunt activity and in the proportion of the population producing superoxide anion. 5-Azacytidine treatment also causes marked inhibition of DNA methyltransferase, and thus DNA synthesized by HL-60 cells during the 24-hr period of analogue treatment is essentially devoid of methylated cytosine residues. This suggests, as does our previous finding that a general inhibitor of transmethylation reactions, l-ethionine, can induce differentiation of HL-60 cells, that changes in gene expression triggered by these compounds may be linked to their ability to alter patterns of DNA methylation. Since at least 50% of HL-60 cells capable of forming colonies in soft agar after a 24-hr exposure to 5-azacytidine yield progeny that mature (i.e., produce superoxide anion in response to 12-O-tetradecanoylphorbol-13-acetate) 2 weeks after 5-azacytidine treatment, the results also indicate that the changes induced in HL-60 cells by limited exposure to 5-azacytidine are heritable and can influence gene expression many generations after treatment has been terminated.