Cellular lipid metabolism can provide a variety of mediators of signal transduction, including diacylglycerols and inositol phosphates. These factors may be involved in the control of epidermal differentiation and proliferation because they are modulated by extracellular calcium, which also regulates the maturation phenotype of cultured keratinocytes. The effect of non-cytotoxic exposures to ultraviolet light on lipid metabolism was studied in cultured murine keratinocytes. Ultraviolet treatment of cultured murine keratinocytes growing in 0.05 mM Ca++ did not significantly change the total amount of [3H]inositol phosphates at 0.5, 8 or 24 h post-irradiation. Irradiated cells responded to an increase from 0.05 mM Ca++ to 1.4 mM Ca++ medium with increased formation of inositol phosphates suggesting irradiation did not alter the normal inositol lipid turnover in response to the Ca++ signal for terminal differentiation. Irradiation (20-120 J/m2 of UVB) induced a dose-dependent increase in the cellular level of diacylglycerols as measured at 24 h post-irradiation, without changing the turnover of other phospholipids including phosphatidylcholine and phosphatidylethanolamine. The increased cellular levels of diacylglycerols following ultraviolet exposure were accompanied by changes in the activity of diacylglycerol kinase (DAG-kinase). The cytosolic DAG-kinase activity was decreased whereas the DAG-kinase activity in the membrane fraction was increased. These results suggest that ultraviolet irradiation increases the level of diacylglycerols via changes in de novo metabolism through a DAG-kinase pathway. Elevated diacylglycerol may influence signal-transduction pathways mediated by cellular lipids and contribute to some keratinocyte responses to ultraviolet light.