The fibronectin content of RMC cultures grown for 8–14 days in medium containing 30 mM (540 mg/dl) D-glucose was increased 30–60% relative to that of control cells cultured in 10 mM (180 mg/dl) glucose. Addition of equiosmolar concentrations (20 mM, 360 mg/dl) of L-glucose, 3-O-methylglucose, or mannitol to 10 mM glucose media did not alter fibronectin accumulation compared with values observed in 10 mM glucose alone. The basal phosphorylation of the 80,000-Mr MARCKS protein, which is a substrate for PKC, and the phosphorylation induced by acute (15-min) exposure of cells to 15% FCS or to 0.1 μM (50 ng/ml) PDBu were all increased in cells grown in 30 mM compared with 10 mM glucose. By contrast, phosphorylation of the 80,000-Mr protein in response to a maximal concentration of PDBu (1 μM, 500 ng/ml) was not different in cells grown in 30 mM compared with 10 mM glucose. The acute increases in phosphorylation of the 80,000-Mr protein were blocked by the PKC inhibitor calphostin C. Chronic (7-day) exposure of mesangial cells grown in 10 mM glucose to 0.1 μM of the PKC agonist PDBu also resulted in a sustained 40% increase in 80,000-Mr phosphorylation and a 20–30% increase in fibronectin accumulation. As assessed by [35S]methionine incorporation, mesangial cell fibronectin synthesis was increased by exposure to PDBu, a finding consistent with earlier observations with 30 mM glucose. The increase in 80,000-Mr proteimesangial cells grown in either 0.1 μM PDBu or 30 mM glucose was abolished in cells whose PKC activity had been downregulated by exposure to PMA. Culture of mesangial cells with the thromboxane-endoperoxide analogue U-46619 in 30 mM, but not 10 mM, glucose increased fibronectin accumulation compared withvalues obtained in 30 mM glucose alone. These results demonstrate a sustained activation of PKC in mesangial cells cultured in a high concentration of glucose and support a role for PKC activation in the mediation of enhanced fibronectin accumulation induced by glucose. Because nonmetabolizable glucose analogues and mannitol were ineffective, metabolism of glucose is implicated in expression of these actions. The results also suggest that a high ambient glucose concentration may interact with thromboxane and/or prostaglandin endoperoxide in the activation of PKC and the enhancement of fibronectin accumulation by mesangial cells.