The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet. Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline. When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level. The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate. The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels. Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased. The methotrexate response in the rats fed the defined choline-deficient diet was different. There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities. The carnitine palmitoyltransferase activity was unchanged. Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet. Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities. In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity. The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.