An increase in glucose concentration in the medium from 5 to 30 mM transiently enhanced diacylglycerol mass and activated protein kinase C in glomeruli isolated from nondiabetic rats as assessed by translocation of enzyme activity from the soluble to participate fraction. Effects of glucose on both diacylglycerol mass and protein kinase C were evident at 5 and 15 min but waned by 30 min. An increase in glucose concentration in the medium also increased the incorporation of [¹⁴C]glucose into the glycerol backbone of diacylglycerol, triacylglycerol, and phospholipids. Several observations implied that [¹⁴C]glucose was being incorporated into diacylglycerol through the de novo pathway for glycerolipid synthesis rather than being derived from phospholipids. 1) [¹⁴C]glucose incorporation into all the lipids was suppressed by 2-deoxyglucose. 2) The incorporation of [¹⁴C] glucose into diacylglycerol and triacylglycerol was evident by 1 min and increased linearly for at least 30 min. In contrast, incorporation into phosphatidylcholine occurred with a lag of at least 5 min. 3) Although only 10% of the [¹⁴C]glucose incorporated into lipids was present in diacylglycerol versus >50% in phospholipids, the specific activity of [¹⁴C]glucose in diacylglycerol was fivefold higher than that in phospholipid when expressed as a function of mass. 4) Glucose had no effect on labeled diacylglycerol or phosphatidic acid production in glomeruli that had been prelabeled with [³H]glycerol. Glucose-induced increases in diacylglycerol may contribute to the activation of glomerular protein kinase C observed in early diabetes.