Methionine has been shown to increase plasma cholesterol in animals. In the present study, mechanisms were investigated by which methionine could alter cholesterol metabolism. In the first experiment, forty growing rats were fed four casein-based diets differing in methionine content (2.6, 3.5, 4.5 or 6.0 g/kg) for 14 d. In the second experiment, isolated rat hepatocytes were incubated in media supplemented with 50, 100 or 200 micromol/l methionine. Dietary methionine tended to increase plasma homocysteine concentrations in the rats (P=0.058). A weak positive correlation between circulating homocysteine and plasma cholesterol was observed (R2 0.27, P<0.01). Rats fed 3.5 g/kg or more of methionine had higher concentrations of cholesterol in their plasma, in lipoprotein fractions of density (rho; kg/l) 1.006<rho<1.063 and rho>1.063, and in liver than rats fed 2.6 g/kg methionine. Rats fed 6 g/kg methionine had a higher hepatic expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol-7alpha-hydroxylase than rats fed less methionine. The phosphatidylcholine:phosphatidylethanolamine ratio in rat liver increased with rising dietary methionine concentration; the relative mRNA concentrations of phosphatidylethanolamine N-methyltransferase and cystathionine beta-synthase remained unaffected. Hepatocytes incubated in media supplemented with 100 or 200 micromol/l methionine had a higher cholesterol synthesis than hepatocytes incubated in a medium supplemented with 50 micromol/l methionine; the LDL uptake in hepatocytes was independent of the methionine concentration of the medium. In conclusion, the present study suggests that dietary methionine induces hypercholesterolaemia at least in part via an enhanced hepatic cholesterol synthesis.