Glyceraldehyde 3-phosphate exists as the geminal diol and the free aldehyde in the molar ratio 29:1 in aqueous solution. The rate constant of the conversion of diol into aldehyde is 8.7x10(-2)sec.(-1) in the pH range 7.3-8.6 at 20 degrees . The free aldehyde is the substrate for d-glyceraldehyde 3-phosphate dehydrogenase. Over a wide concentration range of enzyme the rate of conversion of diol into aldehyde is the rate-limiting process in the catalytic oxidation of d-glyceraldehyde 3-phosphate by NAD(+). Aldolase and triose phosphate isomerase both liberate d-glyceraldehyde 3-phosphate as the aldehyde. This suggests that the relatively slow diol-aldehyde interconversion does not restrict the rate of glycolysis.