AbstractExposure of human sperm to progesterone (P4) activates cation channel of sperm (CatSper) channels, inducing an intracellular Ca2+concentration ([Ca2+]i) transient followed by repetitive [Ca2+]iactivity (oscillations), which are believed to be functionally important. We investigated the potential significance of store-operated Ca2+-entry in these oscillations using the inhibitor SKF96365 (30 µM; SKF). Following pre-treatment of human sperm with 3 µM P4, exposure to SKF doubled the proportion of oscillating cells (P= 0.00004). In non-pre-treated cells, SKF had an effect similar to P4, inducing a [Ca2+]itransient in >80% of cells which was followed by oscillations in ≈50% of cells. The CatSper blocker RU1968 (11 µM) inhibited the SKF-induced [Ca2+]iincrease and reversibly arrested [Ca2+]ioscillations. Using whole-cell patch clamp, we observed that SKF enhanced CatSper currents by 100% within 30 s, but amplitude then decayed to levels below control over the next minute. When cells were stimulated with P4, CatSper currents were stably increased (by 200%). Application of SKF then returned current amplitude to control level or less. When sperm were prepared in medium lacking bovine serum albumin (BSA), both P4 and SKF induced a [Ca2+]itransient in >95% of cells but the ability of SKF to induce oscillations was greatly reduced (P= 0.0009). We conclude that SKF, similar to a range of small organic molecules, activates CatSper channels, but that a secondary blocking action also occurs, which was detected only during patch-clamp recording. The failure of SKF to induce oscillations when cells were prepared without BSA emphasizes that the drug does not fully mimic the actions of P4.