Intracellular pH (pHi) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pHi in these cells are not understood. The pHi of mouse cauda epididymal sperm was determined from the fluorescence excitation ratio of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein and calibrated with nigericin and elevated external [K+]. Two acid efflux mechanisms were identified following imposition of acid loads. One pathway has many anticipated characteristics of the somatic Na(+)-dependent Cl(-)-HCO3- exchanger, although sperm and somatic mechanisms can be distinguished by their ion selectivity and inhibitor sensitivity. Sperm may have an isoform of this exchange pathway with novel functional characteristics. The second acid-export pathway does not require extracellular anions or cations and is inhibited by arylaminobenzoates (flufenamic acid, diphenylamine-2-carboxylate). Mouse sperm also recover spontaneously from intracellular alkalinization. Recovery rates in N-methyl-D-glucamine+ Cl- or in 0.25 M sucrose are not significantly different from that in a complex culture medium. Thus, recovery from alkalinization does not utilize specific, ion-dependent transport mechanisms. Other widely distributed acid-efflux mechanisms, such as the Na(+)-H+ antiport pathway and the Na(+)-independent Cl(-)-HCO3- exchanger are not major regulators of mouse sperm pHi. Sperm capacitation results in pHi increases (from 6.54 +/- 0.08 to 6.73 +/- 0.09) that require a functional Na(+)-, Cl(-)-, and HCO3(-)-dependent acid-efflux pathway. Inhibition of this regulatory mechanism attenuates alkaline shifts in pHi during capacitation as well as the ability of sperm to produce a secretory response to zona pellucida agonists. These data suggest that one aspect of mouse sperm capacitation is the selective activation of one major pHi regulator.