Vitamin D3 treatment of the human promyelocytic cell line, HL-60, is accompanied by an increase in phorbol ester receptor number (Martell, R. E., Simpson, R. U., and Taylor, J. M. (1987) J. Biol. Chem. 262, 5570-5575). In this study, the mechanism and significance of vitamin D3-induced changes in protein kinase C levels were investigated. Treatment of HL-60 cells with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in a 2-3-fold increase in phorbol dibutyrate binding at 24 h. This was accompanied by a 4.2-fold increase in steady state mRNA levels for the beta isoenzyme of protein kinase C and by a 3.8-fold increase in its transcriptional rate as determined from nuclear run-off studies. Protein kinase C alpha mRNA, which was approximately 15% of the beta isoenzyme levels, showed similar increases in mRNA and transcriptional rates in response to 1,25-(OH)2D3. Protein kinase C gamma mRNA was not detected. The increases in protein kinase C levels were accompanied by increases in activation of the enzyme by phorbol esters. More importantly, 1,25-(OH)2D3 caused a 1.5-2.0-fold increase in the endogenous phosphorylation of protein kinase C substrates independent of exogenous activators or endogenous formation of diacylglycerol. This is the first report of transcriptional activation of protein kinase C. This mechanism of up-regulation of protein kinase C may explain the increased activity of protein kinase C in vitamin D3-treated HL-60 cells, and may constitute an important mechanism for the long term modulation of the protein kinase C pathway of cell regulation independent of diacylglycerol generation.