We propose a pathway leading from erythrose-4-phosphate and glutamate to nitrogen 1 and carbons 5,5', and 6 of the pyridoxine ring. This pathway, which parallels the phosphorylated pathway of serine biosynthesis, is predicted on the homology between PdxB and SerA, the structural similarity between serine and 4-hydroxythreonine, and the possible involvement of SerC in pyridoxine biosynthesis. Several predictions of this hypothetical scheme were tested. Consistent with the proposed pathway, supplement inhibition patterns strongly suggest that SerA enzyme acts in a an alternate pathway of pyridoxine biosynthesis in pdxB mutants. Direct enzyme assays detected erythrose-4-phosphate dehydrogenase activity in crude extracts, which again supports the proposed pathway. Chromosomal insertions in serC caused a requirement for pyridoxine, serine, and aromatic compounds, which directly verified that SerC functions in the pyridoxine biosynthetic pathway. Complementation analysis showed that pdxF and pdxC mutations reported previously are most likely alleles of serC. Growth of serC chromosomal insertion mutants on glycoalaldehyde was found to occur without acquisition of second-site mutations and confirmed that pdxB and serC, but not pdxA, function in the same branch of the pyridoxine pathway. In addition, serC::mini-Mu d insertions revealed that the complex serC-aroA operon lacks internal promoters, that the amino terminus of SerC is not strictly essential for activity, and that antisense transcription occurs in the serC-aroA operon. Growth responses of pdxA, pdxB, and serC mutants to beta-hydroxypyruvate, D-alanine, and glycolate could also be reconciled with the proposed pathway. Finally, the proposed scheme is consistent with previous isotope labeling data and accounts for several other observations about pyridoxine biosynthesis. Together, these physiological and biochemical analyses support the proposed pathway and an evolutionary scenario in which this branch of the pyridoxine pathway evolved from the serine pathway by gene recruitment.