© 2019 CSIRO. The mechanism that causes the detachment of spermatozoa from the oviductal reservoir around the time of ovulation remains to be elucidated. Because the cumulus cells of the bovine oocyte are known to secrete progesterone (P4), and P4 has been shown to act upon cation channels of spermatozoa (CatSper) in human spermatozoa, it was hypothesised that P4 could induce hyperactivation due to an influx of extracellular calcium, and this would facilitate detachment of spermatozoa from oviductal epithelial cells. Therefore, this study aimed to investigate the role and mechanism of action of P4 in the release of spermatozoa from bovine oviduct epithelial cells (BOEC). Initial dose-response assessments on sperm hyperactivation determined the optimum concentration of P4 (10 nM), mibefradil (a non-specific Ca 2+ channel antagonist; 5 ¿M), NNC 55-0396 dihydrochloride (NNC; a CatSper antagonist; 2 ¿M), mifepristone (a classical and membrane P4 receptor antagonist; 400 nM) and AG205 (a membrane P4 receptor antagonist; 10 ¿M). BOEC explants were incubated with frozen-thawed bovine spermatozoa for 30 min, following which loosely bound spermatozoa were removed. Two experiments were completed. In Experiment 1, BOECs were treated for 30 min with either no treatment, P4, NNC, mibefradil, P4 + mibefradil, P4 + NNC, P4 + mibefradil + NNC or P4 + EGTA. In Experiment 2, BOECs were treated for 30 min with either no treatment, P4, mifepristone, AG205, mifepristone + AG205, P4 + mifepristone, P4 + AG205 or P4 + mifepristone + AG205. The number of spermatozoa remaining bound per millimetre squared of BOEC explant was determined. Progesterone stimulated the release of bound spermatozoa from BOEC explants, whereas NNC, mibefradil and EGTA inhibited this release. The release of spermatozoa by P4 was inhibited in the presence of both mifepristone and AG205, whereas the combination of both had the greatest inhibitory action on P4 release of spermatozoa. These findings suggest the presence of a P4 membrane receptor on bovine spermatozoa and that P4-induced release of spermatozoa from BOECs is likely mediated by extracellular Ca 2+ .