Earlier studies have shown increased adenylate cyclase (AC) activity in epididymal mouse sperm incubated under capacitating conditions in vitro. The present study investigated the effect on AC activity of excluding calcium and/or glucose from the sperm incubation medium, which would modulate expression of fertilizing potential. AC activity was higher in sperm incubated for 120 than 30 min, and was higher in sperm incubated in calcium-containing than calcium-free media for all except acrosome-reacted populations. Calcium added at the time of assay stimulated AC activity, the degree of this response being independent of the functional state of the sperm population. The guanine nucleotide analogue Gpp(NH)p slightly enhanced AC activity, but did not alter the stimulatory effect of calcium. Since calcium can increase AC activity, possibly by interaction with a divalent cation allosteric site on the catalytic subunit of the enzyme, a rise in intracellular calcium levels during capacitation may mediate the increased activity of AC, allowing expression of cAMP dependent events which are a prerequisite for fertilization.