To obtain further information on the role of the pyridine-nitrogen of vitamin B6 in nonenzymatic and enzymatic systems, the N-oxides of pyridoxal, pyridoxamine, and their 5′-phosphates were synthesized and their catalytic actions studied. In the nonenzymatic α,β-elimination of tryptophan and serine, pyridoxal N-oxide served as a catalyst, though somewhat less efficiently than pyridoxal. Transamination reaction between pyridoxal N-oxide and glutamate occurred only slightly. On the other hand, the reaction between pyridoxamine N-oxide and α-ketoglutarate proceeded readily, but at a slower rate than when pyridoxamine was used. The lower activities of the N-oxides could be ascribed to the decrease in electronegativity of the pyridine-nitrogen. Pyridoxal N-oxide 5′-phosphate served as the coenzyme for E. coli tryptophanase. Although its Km value was nearly equal to that of pyridoxal phosphate, the maximum velocity was only 65% of that of the native coenzyme. In a mammalian glutamic-oxaloacetic transaminase (GOT) system, the activity of the coenzyme analogue-bound apoenzyme varied with the reaction time even after a 10-min preincubation for the reconstitution. In this case, a plot of the reaction velocity vs. coenzyme concentration gave a sigmoidal curve, suggesting a complicated interaction between the coenzyme analogue and the apoenzyme. © 1969.