Pyridoxal kinase from Escherichia coli and bakers' yeast was inactivated by pyridoxal while the enzyme from rat and pig brain was not. The inactivation of the enzyme purified from E. coli was reversible and was rendered irreversible by the reduction with NaBH4. This finding as well as a similar inactivation by 5'-deoxypyridoxal but not by 4'-deoxypyridoxine suggested that the inactivation was due to Schiff base formation. The suggestion was confirmed by the incorporation of tritium label into the enzyme by the reaction of the enzyme with [3H] pyridoxal followed by the treatment with NaBH4. Correlation between the loss of enzyme activity and the amount of pyridoxal bound to the enzyme showed that binding of pyridoxal to one crucial site completely inactivated the enzyme. Pyridoxine and 4'-deoxypyridoxine did not have a protective effect against inactivation indicates that the binding site was not the substrate site. The results of kinetic and equilibrium analyses were consistent with a one-step inactivation mechanism.