Cultured rat hepatocytes were used to examine the rapidly invoked mechanisms through which fatty acids stimulate very low density lipoprotein (VLDL) secretion. Fatty acids (1 mM) bound to albumin (0.25 mM) were added to the serum-free culture medium of hepatocytes. Within 15 min, there was a significant 2-fold increase in the secretion of [3H]triacylglycerol (produced from [3H]glycerol). The stimulation lasted for at least 4 h. For three 18 carbon fatty acids studied, there was an inverse relationship between ability to stimulate triacylglycerol secretion and the number of carbon-carbon double bonds. Since oleic acid caused the greatest stimulation of triacylglycerol secretion, its effects were studied further. Isolation of VLDL from the culture medium showed that oleic acid increased the content of both triacylglycerol (2-3-fold) and phospholipid (38%). However, isolation of a total lipoprotein fraction by both ultracentrifugation (d<1.21 g/ml) and antibody affinity chromatography showed that oleic acid did not affect total apolipoprotein synthesis. When added to hepatocytes obtained from rats having different lipogenic states, oleic acid stimulated [3H]triacylglycerol secretion in the same order as the rate of apolipoprotein synthesis, i.e., sucrose-fed > control > starved. Although oleic acid stimulated [3H]triacylglycerol secretion in all hepatocyte preparations, apolipoprotein synthesis was unaffected. Inhibition of protein synthesis by cycloheximide totally prevented the stimulation of triacylglycerol secretion by oleic acid. Although protein synthesis is required for VLDL triacylglycerol secretion, rapid (4 h) stimulation of triacylglycerol secretion is not accompanied by similar changes in apolipoprotein synthesis. However, rates of apolipoprotein synthesis do play a role in determining the capacity of the hepatocyte to augment triacylglycerol secretion in response to oleic acid.