Sperm hyperactivated motility is characterized by high flagellar bend amplitude and asymmetrical beating, which are detected by computer-assisted sperm motility analysis as increased curvilinear velocity and lateral head movement. It is required for sperm penetration of the oocyte zona pellucida during fertilization and is induced by an increase in flagellar Ca² . Our objective was to determine whether pH plays a role in promoting Ca signaling of hyperactivated motility. The cell-permeant weak base NH₄Cl increased curvilinear velocity and amplitude of lateral head movement of bovine sperm, indicative of hyperactivation. Fluorometric recordings of sperm loaded with BCECF-AM or fluo3-AM, revealed that NH₄Cl evoked elevations of intracellular pH and Ca² , respectively, with the rise in pH occurring more rapidly than that of Ca² . Single-cell image analysis showed increased Ca² levels in the flagellum in response to NH₄Cl. When extracellular Ca² was lowered with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) prior to treatment with NH₄Cl, intracellular pH was increased, but elevation of Ca² and hyperactivation were diminished. This suggests that the rise in intracellular pH precedes an influx of Ca² . The Ca² channel blocker Ni² also diminished NH₄Cl stimulation of hyperactivation, demonstrating that Ca² entry is required for maximal expression of hyperactivation. Ca² ionophore produced an increase in Ca² that was 3-fold greater than that produced by NH₄Cl; however, it produced a weaker hyperactivation response. These results indicate that a rise in pH increases intracellular Ca² and promotes hyperactivation primarily by stimulating Ca² influx, but also by other mechanisms.