Vitamin B-6 vitamer distribution and activities of vitamin B-6 metabolizing enzymes were evaluated in aging male and female Wistar rats fed a purified diet (containing 250 g of casein and 6 mg of pyridoxine hydrochloride per kg) from weaning until 31 mo of age. Plasma pyridoxal 5'-phosphate (PLP) concentration became lower with increasing age, with the largest decrease in the 1st yr of life. An age-related change in vitamin B-6 distribution between the various tissues examined was observed: B-6 vitamer content increased in heart and brain, whereas PLP content decreased in gastrocnemius muscle, kidney and liver. The decrease in muscle PLP content occurred in concert with a decrease in muscle glycogen phosphorylase activity. Urinary 4-pyridoxic acid (4-PA) excretion increased with age, especially in female rats, in parallel with an increase in liver pyridoxal oxidase and pyridoxal dehydrogenase activities. Age-related changes in vitamin B-6 distribution were probably not causally related to changes in activity of vitamin B-6 metabolizing enzymes; they were regarded as consequences of changes in protein metabolism. The higher urinary 4-PA excretion in older rats may reflect a lower vitamin B-6 requirement; however, the lower PLP content of gastrocnemius muscle may indicate an age-related decrease in vitamin B-6 body stores.