Studies reported here determined the effect of dietary fat level on membrane phospholipid composition, phosphoinositide labeling, 1,2-sn-diacylglycerol and protein kinase C activity in epidermal cells from female Sencar mice. Animals were fed either high fat (24.6 g/100 g diet) or control (5 g/100 g diet) diets at constant energy intake for 6 to 7 wk or 15 to 22 wk, and epidermal cells were isolated. The level of phosphatidylinositol was significantly lower in the animals fed the control diet than in the animals fed the high fat diet (0.6 vs. 1.2 nmol/10(6) cells). The fatty acid composition of the phospholipids showed significantly lower arachidonic acid level in phosphatidylinositol when the animals were fed the high fat diet. Protein kinase C activity in the solubilized particulate and soluble fraction of the cells was 131 +/- 18% and 62 +/- 14% greater, respectively, in animals fed the high fat diet compared with animals fed control diet. The level of 1,2-sn-diacylglycerol was significantly higher in animals fed the high fat diet (mean nmol/mg lipid +/- SEM: control, 4.5 +/- 0.5; high fat, 7.0 +/- 0.5). Incorporation of [3H]inositol into inositol lipid was not altered by diet. Because protein kinase C and 1,2-sn-diacylglycerol have been implicated in tumor promotion, the increase in protein kinase C activity and the elevation of 1,2-sn-diacylglycerol in cells from animals fed the high fat diet may be important in the high cancer rate observed with these diets.