Membrane potential (VM) was investigated in mouse and bovine sperm populations. VM was determined from the fluorescence emission of the lipophilic anion, bis(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3)), and from the lipophilic cation, 3,3'-dipropylthiodicarbocyanine iodide (DiSC3(5)). Fluorescent signals were corrected for contributions of mitochondrial potentials and apparent VM values were obtained by calibrations in sperm selectively permeabilized with valinomycin or with gramicidin D. The calculated VM values of uncapacitated mouse and bovine sperm were approximately -35 and -30 mV, respectively. In contrast, capacitated populations of mouse and bovine sperm have VM values of -50 to -60 mV. Membrane hyperpolarization is due in part to an enhanced K+ permeability. The development of zona pellucida-activated signal transducing mechanisms during capacitation is dependent upon hyperpolarization. It is suggested that VM alterations regulate the activation state of sperm, thereby suppressing premature acrosome reactions in uncapacitated sperm and permitting capacitated sperm to respond to zona pellucida stimuli.