In the oxidation of methylglyoxal by 2-oxoaldehyde dehydrogenase, the apparent Km value for NADP+ was about 2.5 times lower than the corresponding Km for NAD+; the apparent Km values for methylglyoxal and for the amine activator L-2-aminopropan-1-ol, with NADP+ as cofactor, were also different from those obtained with NAD+. In the presence of NADP+, the enzyme was not activated by P1, in contrast with the activation of the enzyme when NAD+ was used. The significance of the results is discussed.