Intact fowl spermatozoa became almost immotile at 40 degrees C. In contrast, the presence of 10-1000 nmol calyculin A l-1, a specific inhibitor of protein phosphatase-1 (PP1) and -2A (PP2A), permitted activation of sperm motility in a dose-dependent manner. Calyculin A also stimulated the rate of oxygen consumption by spermatozoa, and induced a concomitant decrease in ATP concentrations, suggesting a coupling of ATP hydrolysis to the rate of oxidative phosphorylation. However, the motility and oxygen consumption of spermatozoa loaded with an intracellular Ca2+ chelator, 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM), were not stimulated by calyculin A alone, but only after the subsequent addition of 2 mmol CaCl2 l-1. These results suggest that inhibition of the activities of endogenous PP1 and PP2A may stimulate the motility and metabolic activity of fowl spermatozoa at 40 degrees C via a mechanism that requires intracellular free Ca2+.