Isolated rat hepatocytes were used to study in vitro effects of 10 mM D-galactosamine (GalN) on hepatic fatty acids metabolism. At this concentration, membrane integrity and biochemical competence (i.e., gluconeogenesis and ureogenesis) remained unaffected. Protein synthesis and secretion, as measured by the incorporation of [U-14C]leucine into total and medium protein, was significantly inhibited when incubated for more than 2 h. GalN activated the incorporation of [U-14C]palmitate into triacylglycerols and depressed its utilization in the formation of labelled ketone bodies and 14CO2. Hepatocytes isolated from fasted rats exposed to GalN in vitro did not show any variation in prelabelled triacylglycerol secretion. GalN induced a rapid inhibition of prelabelled triacylglycerol secretion by hepatocytes isolated from fed rats in which this secretion occurred to a larger extent than in hepatocytes isolated from fasted rats. The data reported here suggest that GalN induces a rise of triacylglycerol synthesis by inhibiting the palmitate oxidation pathway and a decrease of triacylglycerol secretion through an early derangement of the secretory pathway.