Primary cultures of adult rat hepatocytes incubated (24-72 h) in Waymouth's 752/1 medium, exhibited a marked decline (80-90%) in their ability to incorporate 0.5 mM [1,3-14C]glycerol and 1.0 mM palmitate into glycerolipids. The specific activities of glycerol kinase and sn-glycerol-3-P acyltransferase also showed a time-dependent reduction (30-80%). Phosphatidate phosphohydrolase, diacylglycerol cholinephosphotransferase, and fatty acid-CoA ligase activities were unaffected. Insulin and/or glucagon (10(-8) to 10(-6) M) not only prevented these reductions in hepatocyte monolayer glycerolipid formation and enzyme activities but increased (2-5-fold) the level of these processes. Cycloheximide (1 microM) reduced the insulin- and glucagon-dependent increases in sn-glycerol-3-P acyltransferase and glycerol kinase activity and glycerolipid biosynthesis. There was an excellent correlation (r greater than 0.93) between changes in glycerol kinase and sn-glycerol-3-P acyltransferase activity and the capacity of hepatocyte monolayers to incorporate labeled glycerol into glycerolipids under all conditions studied. Therefore, insulin and glucagon may regulate hepatic glycerolipid biosynthesis in part by maintaining the liver cell's enzymatic rate of glycerolipid biosynthesis.