Physiologic and pharmacologic factors affecting intracellular red cell vitamin B(6)metabolism in normal human subjects were studied using a new assay for pyridoxine kinase (PnK)together with saturated and total aspartate aminotransferase (AST) activities as indirect indices of intracellular pyridoxal 5-phosphate (PLP) availability. The presence of reduced PnK activity in Blacks was confirmed but this could not be explained on the basis of increased enzyme inactivation during red cell aging in vivo. Racial differences were also noted in the metabolism of AST and,in Caucasians, net dissociation of PLP from the apoprotein was demonstrated to occur in vivo. Despite the wide variation in PnK activity, AST levels were maintained within relatively narrow limits. However, when pharmacologic doses of pyridoxine were administered, PnK and AST activities increased proportionately. These findings suggest that when the supply of B6 vitamers is not limiting, PnK may play a role in regulating red cell PLP levels.