The origin of the nitrogen atom of pyridoxine was studied in Saccharomyces cerevisiae. The 15N atom of 15NH4Cl added to the growth medium as the nitrogen source was incorporated efficiently into the nitrogen atom of pyridoxine. The competitive effects of 14N-amino acids on the incorporation of 15NH4Cl were examined. Incorporation of 15N into pyridoxine was inhibited by glutamine. The label of L-[amide-15N]glutamine was incorporated effectively into pyridoxine in S. cerevisiae. On the other hand, the label from L-[amide-15N]glutamine was not incorporated into pyridoxine in Escherichia coli. These findings suggest that the biosynthetic pathway of pyridoxine in S. cerevisiae differs from that in E. coli.