A method was developed to isolate flagella with intact membranes from sea urchin sperm to further study the mechanism of the Na+-dependent H+ extrusion which increases the intracellular pH and triggers motility. Based on cytochrome c oxidase activity and cell count, the flagellar preparation contained at most 0.05% of the sperm heads present in the initial homogenate. The isolated flagella retained a Na+-dependent H+ extrusion mechanism which, like that of intact sperm, was inhibitable by high external potassium and with an ionic selectivity in the order of Na+ greater than Li+ much greater than Rb+ approximately equal to Cs+ (essentially ineffective). Two methods were employed to measure the change in intraflagellar pH (pHi): the accumulation of [14C]methylamine and a spectrofluorimetric technique using acridine orange. Flagella isolated from sperm suspended in Na+-free seawater (pH 7.90) maintained a pHi of 6.72 +/- 0.05 which increased by 0.5 to 0.6 pH unit following a Na+ (10 mM)-dependent release of H+ (114 +/- 18 nmol/mg) and an uptake of 22Na+ (134 +/- 36 nmol/mg). Both ionic movements were inhibitable by high external K+, suggesting a coupled exchange mechanism. This study provides direct evidence for the alkalinization of the flagellar pH under a condition compatible with motility initiation and shows that the isolated flagella is a useful model system for studying its mechanism.