1. Liver slices from chronically alcohol-fed rats incubated with 3H2O showed less than half the fatty acid synthesis rates of pair-fed controls. Addition of 50 mmol/l ethanol or of 10 mmol/l lactate and 1 mmol/l pyruvate to the incubation medium did not alter the fatty acid synthesis rates in either groups. Hepatic fatty acid synthesis rates measured in vivo with 3H2O were also significantly reduced in alcohol-fed rats. 2. Time-course experiments showed that after 1 week on the ethanol diet hepatic fatty acid synthesis rates in vitro were similar to control rats, although the liver triacylglycerol content was significantly increased. From the second week of feeding, fatty acid synthesis rates were significantly lower in alcohol-fed rats and the liver triacylglycerol content progressively increased compared with controls. 3. Fatty acid synthase activity in liver cytosolic fractions were similar to controls in the alcohol-fed group after 1 week of feeding but were significantly lower in alcohol-fed rats from the second week onwards. 4. These results indicate that hepatic triacylglycerol accumulation after alcohol feeding is not due to increased fatty acid synthesis. The reduced fatty acid synthesis observed is a consequence of triacylglycerol accumulation.