Recently developed flow cytometric methods (Trón et al.: Mol Immunol 27:1307-1311, 1990) to measure the intracellular pH (pHi) and intracellular potassium concentration in mammalian cells by using the fluorescent pH-indicator dye 2',7bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF) were adopted for measuring these parameters in carp sperm. The intracellular potassium concentration of the carp sperm was 62.4 +/- 5.3 mM. This is very similar to the potassium concentration of the seminal plasma (87 +/- 16 mM), and it suggests a depolarized state of the sperm cell in the semen. An average pHi value of 7.06 +/- 0.11 was obtained by measuring sperm samples taken from ten animals. Changes in the ionic composition of the environment did not alter pHi. Sperm motility was initiated by transferring the cells to an environment of 110 mOsm osmolality. This hypoosmotic shock induced fast changes in the membrane structure that could be reversed by restoring physiologic osmolality. Activation was accompanied by a fast alkalinization of the sperm cells. This pH change was amiloride sensitive, suggesting the involvement of the Na+/H+ exchanger in the activation process. Alkalinization of acid-loaded sperm cells depended on the osmolality of the environment. Equilibrium pHi of these cells in hyperosmotic buffers was substantially lower relative to cells in an isoosmotic environment. Effects of the extracellular and intracellular pH on carp sperm motility were also examined. Extracellular pH below 5.5 abolished sperm motility completely. Alkaline extracellular pH did not alter the duration of sperm motility even at extreme values (pHe = 9.6). Duration of the flagellar motion did not depend on the pHi between values of 6.5 and 8.5; however, it was significantly reduced both below and above this range. No motility was observed below pHi = 6.0 or above pHi = 9.5 with a 10 min incubation time at these pH values prior to activation. Effects of the extracellular and intracellular pH on sperm motility were partially reversible.