Nitric oxide (NO) or NO-generating compounds like sodium nitroprusside (SNP) increase cellular levels of cGMP and produce S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. In search of a reagent that could discriminate between these two effects, we used the sesquiterpene antibiotic koningic acid, which binds to GAPDH at the Cys-149 of the active site. Koningic acid inhibited basal and sodium nitroprusside-stimulated NAD-dependent covalent modification of purified rabbit muscle GAPDH in a dose-dependent manner. Furthermore, we tested the effect of koningic acid on human platelets. Approximately 90% of GAPDH is present in the cytosol of human platelets, and the exposure of platelet cytosol to koningic acid inhibited GAPDH activity, while the soluble guanylyl cyclase (basal and sodium nitroprusside-stimulated) activity remained unaltered. Pretreatment of intact platelets with koningic acid slowed the rate of aggregation induced by a submaximal concentration of thrombin. In addition, the antibiotic also inhibited the cGMP increases triggered by SNP, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndomidine (SIN-1) but failed to prevent an increase in cGMP caused by nitrosylated albumin. Under the same conditions, koningic acid also inhibited basal and SNP- SNAP-, and SIN-1-stimulated NAD-dependent modification of GAPDH and its enzymatic activity. These results suggest that the mechanism of delivery of NO from SNP, SNAP, and SIN-1 to platelets may require the active form of GAPDH. When NO is delivered by nitrosylated albumin, active GAPDH was not necessary.