Capacitation of hamster caudal spermatozoa at a density of 1 x 10(6)/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation. Treatment of spermatozoa at a density of 1 x 10(6)/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility. To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors. These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species.