Abstract Gossypol was isolated and purified from cotton seed flour. It was found to inhibit rat testis cytosolic LDH-X activity, in vitro , in a dose-dependent manner. Preincubation of the enzyme with gossypol increased the inhibitory effect markedly. Addition of NADH to the preincubation mixture imparted some protection against inhibition. The inhibitory effect of gossypol was competitive with respect to NADH, but non-competitive with respect to α-ketoglutarate. The latter is reported to be a specific substrate for rat LDH-X and hence can be used for measuring LDH-X activity in the presence of other lactic dehydrogenase isoenzymes. Preliminary studies show that gossypol can inhibit other dehydrogenases such as glutamic dehydrogenase, glutathione reductase and malic dehydrogenase as well. The antifertility effect of gossypol in males may be mediated through its inhibitory effect on dehydrogenase enzymes, particularly LDH-X.