The effect of different nucleotides and glycolytic intermediates was tested on the NAD-linked and NADP-linked alpha-ketoaldehyde dehydrogenases involved in the oxidation of methylglyoxal to pyruvate. ATP, GTP, and ADP strongly inhibit the NAD-linked enzyme whereas activity of the NADP-linked enzyme remained unaltered. NADP at a concentration much below its catalytic concentration strongly inhibited the NAD-linked enzyme. This NADP inhibition decreased with decreasing of the pH of the incubation medium. Fructose 1,6-bisphosphate stimulated and glyceraldehyde 3-phosphate inhibited the activity of the NAD-linked enzyme whereas dihydroxyacetone phosphate inhibited NADP-linked activity. The stimulatory effect of fructose 1,6-bisphosphate diminished with lowering of the pH value. The various effects by several key metabolites of the glycolytic pathway indicate a possible physiological role for these enzymes.