HL 60 cells were subcloned into variants with different sensitivities to the differentiation-inducing effect of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), as shown by increased oxidative and phagocytic activity and the appearance of cytoplasmic alpha-naphthyl butyrate esterase. Brief treatment (4 h) with 1,25(OH)2D3 or 5-azacytidine (5-Aza CR) of sensitive sublines induced monocytic differentiation in a fraction of treated cells. The phenotypic differentiation induced by 1,25(OH)2D3 was preceded by altered expression of oncogenes c-myc and c-fos, but not of c-Ha-ras or of the constitutively expressed p72 gene. Treatment with 5-Aza CR had similar effects but in addition increased the rate of transcription of the c-Ha-ras oncogene. In partially resistant sublines exposure to 1,25(OH)2D3 or 5-Aza CR resulted in changes in the levels of c-myc and c-fos mRNA, but the expression of c-Ha-ras gene did not correlate with the phenotypic differentiation. When induction of differentiation in responsive cells was delayed by cycloheximide, a temporal correlation could be made between changes in the expression of the c-myc gene and differentiation. These results suggest that a reduction of the elevated levels of c-myc gene transcription in HL 60 cells is required for the initiation of monocytic differentiation, that the induction of c-fos gene expression is an early step in this process, and that the transient increase in the expression of c-Ha-ras gene by 5-Aza CR is not related to differentiation.