Exposure of HL-60 promyelocytic leukemia cells to calcitriol results in a decrease in steady-state levels of c-myc mRNA and induces cellular differentiation. We have asked whether calcitriol has a direct effect on the transcription of the c-myc gene. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) decreased RNA elongation in a nuclear run-off transcription assay by 4 h after treatment. In the continuous presence of 1,25-(OH)2D3, HL-60 cell transcription of c-myc was decreased by 38% at 4 h and was abolished by 48 h. In contrast, the transcription of beta-actin was not affected by 1,25-(OH)2D3 treatment. The rate of transcription of c-myc and beta-actin was proportional to the number of nuclei and to time. Furthermore, specific hybridization of c-myc and beta-actin RNA was a linear function of RNA input. After a 48-h treatment, the c-myc/beta-actin ratio was decreased by 80-100% at [32P]RNA inputs ranging from 2 to 20 X 10(6) cpm/ml. These data temporally correlate inhibition of c-myc transcription with decreases in the steady-state levels of c-myc mRNA as assessed by Northern blot analysis. We conclude that the effect of 1,25-(OH)2D3 on c-myc expression occurs at the transcriptional level.