S-adenosylmethionine, formed by the adenylation of methionine via S-adenosylmethionine synthase, is the methyl donor in virtually all known biological methylations. These methylation reactions produce a methylated substrate and S-adenosylhomocysteine, which is subsequently metabolized to homocysteine. The methylation of guanidinoacetate to form creatine consumes more methyl groups than all other methylation reactions combined. Therefore, we examined the effects of increased or decreased methyl demand by these physiological substrates on plasma homocysteine by feeding rats guanidinoacetate- or creatine-supplemented diets for 2 wk. Plasma homocysteine was significantly increased (~50%) in rats maintained on guanidinoacetate-supplemented diets, whereas rats maintained on creatine-supplemented diets exhibited a significantly lower (~25%) plasma homocysteine level. Plasma creatine and muscle total creatine were significantly increased in rats fed the creatine-supplemented or guanidinoacetate-supplemented diets. The activity of kidney L-arginine:glycine amidinotransferase, the enzyme catalyzing the synthesis of guanidinoacetate, was significantly decreased in both supplementation groups. To examine the role of the liver in mediating these changes in plasma homocysteine, isolated rat hepatocytes were incubated with methionine in the presence and absence of guanidinoacetate and creatine, and homocysteine export was measured. Homocysteine export was significantly increased in the presence of guanidinoacetate. Creatine, however, was without effect. These results suggest that homocysteine metabolism is sensitive to methylation demand imposed by physiological substrates.