To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.