This prospective study assesses the effect of 2.5, 4, and 10 mg of pyridoxine supplementation during pregnancy on maternal and fetal plasma levels of pyridoxal 5'-phosphate (PLP) and on the degree of coenzyme saturation (activation factor) of aspartate aminotransferase and alanine aminotransferase (alphaEGOT and alphaEGPT) in maternal erythrocytes. More than 4 mg of pyridoxine supplementation daily was required for most pregnancies to maintain maternal plasma PLP levels within the range observed during the first trimester and in the nonpregnant state. The plasma PLP concentrations in maternal and cord blood were highly correlated and indicated a dependence of fetal vitamin B6 nutrition on maternal circulating PLP. Measurements of alphaEGOT and alphaEGPT were not as reproducible as plasma PLP assays and were less sensitive and quantitative indicators. In the majority of subjects, the changes in alphaEGOT and alphaEGPT with time correlated poorly with the changes in plasma PLP. However, when the data were analyzed without regard for their dependence on time, they demonstrated a negative, linear correlation between alphaEGOT and log plasma PLP and between alphaEGPT and log plasma PLP for the group on 2.5 mg of pyridoxine and for all the subjects combined. Finally, the dietary records showed that most of the subjects consumed less than 2 mg of vitamin B6 daily from their food. The results indicate that the current Recommended Dietary Allowance for vitamin B6 during pregnancy (2.5 mg) is too low and that supplementation of this vitamin in an amount more than 4 mg daily is recommended.