Image analysis techniques have been used to demonstrate that progesterone induces a rapid calcium transient in the acrosomal domain of greater than 90% of human spermatozoa (n = 2354). These results are at variance with previous reports, suggesting that progesterone receptors are only expressed on a small subpopulation of these cells, by virtue of their ability to bind fluorescent probes incorporating progesterone 3- (O-carboxymethyl) oxime conjugated to BSA. In the present study, we could confirm that such probes only bound to a small proportion of human spermatozoa (3.01 +/- 0.29%; n = 7557) although 91.79 +/- 1.8% of the same sperm populations exhibited a calcium transient in response to progesterone. These results indicate that the binding of labeled progesterone conjugates to human spermatozoa does not reflect the size of the progesterone responsive population; the response elicited by this steroid is essentially ubiquitous. Progesterone action was shown to involve an influx of extracellular calcium via mechanisms that did not involve voltage sensitive- or second messenger operated-channels, phospholipase C, or G proteins. Despite previous evidence suggesting that progesterone action might involve a GABAA receptor/chloride channel, neither GABA nor the GABA agonist muscimol had any effect on intracellular calcium concentrations in human spermatozoa or influenced their functional competence. The only factor that disrupted the responses of human spermatozoa to progesterone was this steroid itself. Progesterone exposure induced a prolonged period of refractoriness to further stimulation that influenced the capacity of these cells to generate calcium transients, and their ability to exhibit a biological response to changes in intracellular calcium. There are implications in these results for our understanding of the extragenomic action of progesterone on human spermatozoa and the clinical manipulation of this system for the assessment and suppression of human sperm function.