Sperm cytosolic pH, determined by the spectral properties of intracellular carboxyfluorescein, is decreased rapidly by the diffusion and subsequent dissociation of the uncharged weak acids pyruvic, lactic, or hydroxybutyric and is increased by diffusion and subsequent intracellular protonation of the weak base NH3. Metabolic and kinetic activity increases dramatically when intracellular pH is elevated above 6.8-6.9 by addition of 50 mM NH4Cl to sperm suspended in a 120 mM NaCl medium. Respiratory stimulation is not observed upon comparable additions of 50 mM Li+ or K+ or when the pH of the medium is increased from 6.5 to 8.2. However, increases of the external pH to 7.8-8.2 in medium employing 120 mM KCl result in increased metabolic and kinetic activity, comparable to the maximal stimulation induced by the phosphodiesterase inhibitor caffeine. An increase in cytosolic pH from 6.3-6.6 to 6.8 occurs concomitant with the respiratory stimulation induced by KCl in alkaline media. No change in cytosolic pH follows addition of caffeine. Cyclic AMP-dependent protein kinase activity ratios, determined in cellular extracts, are increased by caffeine treatment but are not elevated by 120 mM KCl, by alkaline pH, or by their combination. These observations indicate that cytosolic pH plays a role in the regulation of motility and metabolism of mammalian sperm that is not mediated by cyclic AMP but that may be under control of a plasma membrane voltage-dependent proton channel. However, H+ fluxes across vesicles prepared from sperm membranes are unaffected by variation in the magnitude of the transvesicular K+ concentration gradient.