Experiments were conducted to clarify the relationship between dietary protein level and plasma homocysteine concentration in rats. Male Wistar rats were fed diets differing in casein level from 5 to 50% for 14 d (Expt. 1). Plasma total homocysteine concentration was positively correlated with dietary casein level in the range of 5 to 10% but inversely correlated with dietary casein level in the range of 10 to 50%. Hepatic cystathionine β-synthase (CBS) and betaine-homocysteine S-methyltransferase (BHMT) activities and renal CBS activity increased in response to dietary casein level in the range of 10 to 50%, whereas hepatic serine and betaine concentrations decreased with increasing dietary casein levels. When rats were fed the 10% casein diet or 10% casein+17.2% amino acid mixture diet for 14 d, plasma homocysteine concentration was significantly lower in rats fed the amino acid mixture-added diet than in rats fed the 10% casein diet (Expt. 2), indicating that the hypohomocysteinemic effect of high casein diets was elicited by amino acids, not by casein contaminants. The degree of increase in plasma homocysteine concentration caused by dietary supplementation with 0.75% L-methionine was significantly lower in rats fed the 40% casein diet than in rats fed the 10% casein diet (Expt. 3). These results indicate that high casein diets do not increase but rather decrease plasma homocysteine concentration and cause resistance to hyperhomocysteinemic treatment, and suggest that such effects of high casein diets are mediated at least by increased activities of CBS and BHMT.