The amide nitrogen atom of glutamine was incorporated into pyridoxine in four eukaryotes, Emericella nidulans, Mucor racemosus, Neurospora crassa and Saccharomyces cerevisiae, and two prokaryotes, Staphylococcus aureus and Bacillus subtilis, but not in the following prokaryotes, Pseudomonas putida, Enterobacter aerogenes and Escherichia coli. On the other hand, the nitrogen atom of glutamate was incorporated into pyridoxine in P. putida, E. aerogenes and E. coli, but not in S. aureus and B. subtilis. These results suggest that there are at least two different biosynthetic routes for pyridoxine and the difference does not depend on prokaryotes and eukaryotes.