The transsulfuration pathway, which aids in regulating homocysteine concentration and mediates cysteine synthesis, may be sensitive to vitamin B-6 status because cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL) require pyridoxal 5'-phosphate (PLP). To assess relations between vitamin B-6 and transsulfuration, we evaluated the effects of dietary pyridoxine (PN) on the hepatic concentration of relevant metabolites and in vitro activity of CBS and CGL. Growing rats were fed AIN-93G- or AIN-76A-based diets that ranged from adequate to deficient in vitamin B-6 (2, 1, 0.5, 0.1, or 0 mg of PN/kg diet, n = 5). This design allowed assessment of the effects of supplemental methionine (AIN-76A) vs. cysteine (AIN-93G) in common research diets over a range of vitamin B-6 levels. CBS activity, assayed in the presence or absence of added S-adenosylmethionine, was independent of diet type and PN level. CGL activity was independent of diet type but proportional to dietary PN. Rats fed deficient (0 and 0.1 mg PN/kg) diets exhibited only approximately 30% of the CGL activity of those fed the 2 mg PN/kg diets. Hepatic cystathionine increased from 20 to 30 nmol/g for the 1-2 mg PN/kg diets to approximately 85 nmol/g for the 0 mg PN/kg diet; however, cysteine was reduced only in B-6-deficient rats consuming the AIN-93G diet (means of 30-40 nmol/g for adequate to 11.6 nmol/g for 0 mg PN/kg AIN-76A diet). In spite of these effects, hepatic glutathione concentration increased in vitamin B-6 deficiency. These results suggest that vitamin B-6-dependent changes in transsulfuration do not limit hepatic glutathione production.