From the modification of the lysine residues of yeast and sturgeon muscle glyceraldehyde 3-phosphate dehydrogenase by 2,4,6-trinitrobenzene sulphonate the following results are obtained. 1. (a) In the absence of NAD + 1.1. (i) The trinitrophenylation follows a biphasic process. During the first phase two and four lysine residues are modified with a loss of 50% and 80% of the dehydrogenase activity in sturgeon muscle enzyme and yeast enzyme, respectively. 1.2. (ii) The modification of these more reactive lysine residues has very little effect on the K m of d-glyceraldehyde 3-phosphate, NAD + or arsenate; only V m is decreased; however, the esterase activity measured with p-nitrophenol acetate remains at about 85% of the initial activity. 1.3. (iii) The trinitrophenylation of these lysine residues in the yeast enzyme abolishes almost totally the S → N acetyl shift from cysteine 149 to lysine 183, whereas in the muscle apoenzymes it is partially prevented. 2. (b) In the presence of NAD + 2.1. (i) With the sturgeon muscle enzyme the trinitrophenylation is a first-order reaction and the loss of activity follows a first-order process, results which are similar to those obtained with the rabbit muscle enzyme (Foucault et al., 1974). Moreover, the rate of trinitrophenylation of lysine residues is identical to the rate of trinitrophenylation of the “slow” reacting lysine residues of apoenzyme, thus indicating that only the most reactive lysine residues are protected by the conformational change induced in the protein by the coenzyme. 2.2. (ii) With the yeast enzyme, trinitrophenylation of the holoenzyme still follows a bipliasic process; however, both rates are decreased as compared with the apoenzyme. The conformational changes induced by NAD + affect the reactivity of both classes of lysine residues. 3. (c) From the common effects of the trinitrophenylation of the most reactive lysine residues on the enzyme activity of the three dehydrogenases, as well as on the specific S → N acetyl transfer with acetylphosphate, it seems that the same lysine residues are being modified and that these might be either lysine 183 or lysine 191, which is located in the proximity. 4. (d) The difference in the number of lysine residues that are trinitrophenylated in the first phase of the chemical modification of the yeast and muscle enzymes is related either to the geometrical disposition of the subunits in the oligomeric structure or to the distance between the active centre regions which are face to face in the dimers (Buehner et al., 1974).