The ability of compounds releasing nitric oxide (NO) to regulate glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH) activity was analysed both in cell homogenates and in intact Dictyostelium discoideum. The time course of GraPDH inactivation in cell lysates by NO‐releasing compounds suggests that two processes may be involved, one of which accounts for the majority of the inactivation and shows a close correlation with GraPDH ADP‐ribosylation. Maximal ADP‐ribosylation under these conditions exhibited a stoichiometry of about 0.4 mol ADP‐ribose/mol enzyme tetramer. NO‐mediated inhibition of GraPDH activity was attenuated if specific substrates, cofactors, or cysteine were added to cytosol preparations. Under such conditions, ADP‐ribosylation of the enzyme was correspondingly reduced or negligible. Intact cells treated with NO‐releasing compounds were shown to respond by rapidly decreasing their GraPDH activity. This inhibition was transient and, after a 10‐min incubation, enzyme activity returned to the level seen in control cells. The time course of these in vivo changes correlated well with those of the NO‐stimulated ADP‐ribosylation of GraPDH also seen in intact cells. The basis underlying the NO‐stimulated inhibition of GraPDH activity was investigated and found to reflect a decreased V_max+ No changes in either the K_m of the enzyme for its substrates or its state of polymerization were observed.