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Whilst fertilizing capacity depends upon a K+ conductance G? that allows the spermatozoon membrane potential Vm to be held at a negative value, the characteristics of this conductance in human sperm are virtually unknown. We therefore studied the biophysical / pharmacological properties of the K+conductance in spermatozoa from normal donors held under voltage / current clamp in the whole cell recording configuration. Our standard recording conditions were designed to maintain quasi-physiological, Na+, K+ and Cl+ gradients. Experiments that explored the effects of ionic substitution / ion channel blockers upon membrane current / potential showed that resting Vm was dependent upon a hyperpolarizing K+ current that flowed via channels that displayed only weak voltage dependence and limited (~7 fold) K+ versus Na+selectivity. This conductance was blocked by quinidine (0.3 mM), bupivacaine (3 mM) and clofilium (50 µM), NNC55-0396 (2 µM) and mibefradil (30 µM), but not by 4-aminopyridine (2 mM, 4-AP). Progesterone had no effect upon the hyperpolarizing K+ current. Repolarization after a test depolarization consistently evoked a transient inward "tail current" (/Tail) that flowed via a second population of ion channels with poor (~3 fold) K+ versus Na+ selectivity. The activity of these channels was increased by quinidine, 4-AP and progesterone. Vm in human sperm is therefore dependent upon a hyperpolarizing K+ current that flows via channels that most closely resemble those encoded by Slo3. Although 0.5 µM progesterone had no effect upon these channels, this hormone did activate the pharmacologically-distinct channels that mediate ITail. In conclusion, this study reveals three functionally and pharmacologically distinct cation channels, Ik, ITail, ICatSper.