Abstract: 1) Extracts of rabbit liver and brain were found to catalyze the conversion of pyridoxine to pyridoxal phosphate in the presence of ATP. The oxidation of pyridoxine to pyridoxal was also observed in liver extract. 2) The same liver extract was shown to convert the synthetic pyridoxine phosphate to pyridoxal phosphate. 3) It was established that the oxidation of pyridoxine to pyridoxal and that of pyridoxine phosphate to pyridoxal phosphate were carried out by two distinct enzymes, which were separated from each other by the use of ethanol fractionation and calcium phosphate gel treatment. 4) A direct spectrophotometric evidence for pyridoxal phosphate formation was presented. 5) From the comparison of two activities in liver extract, it was suggested that pyridoxine ingested would be converted to pyridoxal phosphate according to the following sequence: pyridoxine → pyridoxine phosphate → pyridoxal phosphate.