Although ion channels are known to be pivotal to sperm function, the technical difficulty of applying electrophysiological techniques to spermatozoa has prevented significant progress in this area. This is due to the cell size and angular shape in combination with their motility. Using a refined technique, specifically for patch clamping spermatozoa, we have made recordings from human cells. This technique permitted approaches which enable functional analysis of sperm ion channels, including acquisition of inside-out patches, generation of averaged currents, and observation of the effects of pharmacological manipulation during prolonged recordings. As well as a low conductance (7 pS) channel and a 25-pS channel, the most striking finding was the presence of very high conductance, 4-aminopyridine-sensitive multistate channels resembling the non-selective cation channel of sea urchin and mouse spermatozoa. Application of 2 mM 4-aminopyridine (a dose sufficient to cause channel blockade) caused an instant and dramatic transition of motility in the sperm population increasing hyperactivated motility by more than 10-fold as assessed by computer-assisted semen analysis. Combined application of patch clamp and pharmacological investigation of mature spenn cells and will permit rapid advances in our understanding the role of ion channels in sperm function. (C) 2004 Elsevier Inc. All rights reserved.