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We show that a tktA tktB double mutant, which is devoid of the two known transketolase isoenzymes of Escherichia coli K-12, requires pyridoxine (vitamin B6) as well as the aromatic amino acids and vitamins for growth. This pyridoxine requirement can also be satisfied by 4-hydroxy-L-threonine or glycolaldehyde. These results provide direct evidence that D-erythrose-4-phosphate is a precursor of the pyridine ring of pyridoxine. In addition, they show that the two major E. coli transketolase isoenzymes are not required for the biosynthesis of D-1-deoxyxylulose, which is thought to be another precursor of pyridoxine.