The kinetics of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with 5,5'-dithiobis-(2-nitrobenzoic acid) show that NAD(+) dissociates from the enzyme before the reaction. In contrast 2-chloromercuri-4-nitrophenol reacts with the holoenzyme without prior dissociation of NAD(+). These studies and observations on the dissociation constant of NAD(+) to the lobster enzyme show that NAD(+) must dissociate from sites modified by substrates during the reductive dephosphorylation of 1,3-diphosphoglycerate. All four sites per tetramer of the apoenzyme are acylated by 1,3-diphosphoglycerate. Hydrolysis of the acyl-enzyme occurs at a significant rate even in the absence of NAD(+), which may explain previous estimates that only two sites per tetramer can readily be acylated.