Ca signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca signalling, stimulates a biphasic [Ca] rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca ] transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck. Pre-treatment with 5 µM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] 'amplified' progesterone-induced [Ca] transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca] response was unchanged. 2-APB (5 µM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca accumulation downstream of the potentiated [Ca] transient. Pre-treatment with 50-100 µM 2-APB failed to potentiate the transient and suppressed sustained [Ca] elevation. When applied during the [Ca] plateau, 50-100 µM 2-APB caused a transient fall in [Ca], which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca ] signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pH (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca] elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca] signals of human sperm.