The (S)-isomer of the male antifertility agent alpha-chlorohydrin strongly inhibited the oxidative metabolism of fructose by boar spermatozoa in vitro. The result of this action, which has been deduced to be an inhibition of glyceraldehydephosphate dehydrogenase, caused an accumulation of fructose-1,6-bisphosphate and the triosephosphates, and a decrease in substrate-level phosphorylation with a concomitant lowering of the energy charge potential of the spermatozoa. The (R)-isomer of alpha-chlorohydrin had no inhibitory activity on fructolysis. A study of the comparative metabolism of (R)-[3-36Cl]-alpha-chlorohydrin and (R,S)-[3-36Cl]-alpha-chlorohydrin by boar spermatozoa showed that it is the (S)-isomer that specifically undergoes a process of oxidative metabolism to (R)-3-chlorolactaldehyde. It is proposed that this endogenous oxidation product, which has the same absolute configuration as the substrate for glyceraldehyde-phosphate dehydrogenase, is the active metabolite of (S)-alpha-chlorohydrin that inhibits this enzyme. Exogenous (R,S)-3-chlorolactaldehyde inhibited the oxidative metabolism of fructose by boar spermatozoa, apparently by a mechanism similar to that of (S)-alpha-chlorohydrin.