We examined the mechanisms that control the downregulation of the c-myc mRNA during differentiation of HL60 cells. On treatment with dimethyl sulfoxide, HL60 cells downmodulated their steady-state c-myc message levels, ceased to proliferate, and underwent terminal differentiation. In nuclear run-on assays in which distinct segments of the c-myc gene were used as probes, an increased blocking to elongation of nascent c-myc transcripts was shown during the early phase of differentiation. During a later phase, however, a loss of transcriptional initiation was observed. This loss of promoter activity correlated well with dramatic changes in the chromatin structure of the c-myc gene, as determined by DNase I-hypersensitive site analysis. In particular, two hypersensitive sites near the two major c-myc promoters disappeared at the time that promotion abated. The newly described, later-acting negative transcriptional control of c-myc also correlated temporally with the inability to reverse the downregulation of the c-myc message quickly on withdrawal of the differentiating agent. Therefore, a terminal step during differentiation may be linked to the later-acting mode of transcriptional regulation of c-myc. The evidence presented in this report has implications for tumorigenesis in Burkitt lymphomas, in which the germ line, nontranslocated c-myc allele is transcriptionally silent.