We have investigated the reversibility of biochemical and physiological changes that occur upon suspension of ejaculated human spermatozoa during in vitro capacitation. Cells were swum up in a simple HEPES-based saline [lacking bicarbonate and bovine serum albumin (BSA)], then resuspended either in supplemented Earle's balanced salt solution (sEBSS) (25 mM bicarbonate) with 0.3% BSA (for in vitro capacitation) or in medium-lacking bicarbonate and/or BSA. Progesterone-induced acrosome reaction (AR) developed during in vitro capacitation (6 h). A progesterone-induced [Ca2+]i signal was detectable in cells maintained in the simple HEPES-based saline, but upon transfer to sEBSS, the response increased three- to four-fold, saturating within <30 min. Serine/threonine phosphorylation saturated within minutes of resuspension, but tyrosine phosphorylation developed over 3 h. Return of cells to non-capacitating conditions caused reversal of all capacitation-dependent changes. The [Ca2+]i signal reverted to its 'uncapacitated' size within <30 min. Protein phosphorylation reversed gradually and could be reinduced (kinetics resembling the first response) upon resuspension in sEBSS. The ability of cells to undergo progesterone-induced AR fell to levels similar to those in uncapacitated cells within 1 h of resuspension in medium not supporting capacitation. Loss of protein phosphorylation occurred only in the absence of both bicarbonate and BSA, but effects on [Ca2+]i signalling and AR could be seen after removal of only one of these factors. We conclude that key events in the capacitation of human spermatozoa are both reversible and repeatable.