At some time before fertilization, mammalian sperm undergo a change in movement pattern, termed hyperactivation. There is evidence that hyperactivation offers an advantage to sperm for detaching from the oviductal mucosa, for penetrating viscoelastic substances in the oviduct, and for penetrating the zona pellucida. Hyperactivation is known to require extracellular calcium, but little else is known about the mechanisms by which calcium affects sperm movement. The calcium-sensitive fluorescent dye indo-1 was used to follow intracellular calcium levels ([Ca2+]i) in individual moving sperm. Sperm were loaded with 10 microM of the acetoxymethyl ester form of the dye and then rinsed. The dye was excited at 340 nm by using a filtered xenon stroboscope, and images at the 405-nm and 490-nm excitation maxima were simultaneously digitized at 30 per sec for 2.1 sec. [Ca2+]i was significantly higher in the acrosomal and postacrosomal regions of the head and in the flagellar midpiece (the principal piece could not be measured) in hyperactivated than in nonhyperactivated sperm (P < 0.0001). [Ca2+]i oscillations were detected in the proximal half of the midpiece that were identical in frequency to the flagellar-beat-cycle frequency in 12 of 17 hyperactivated sperm (median, 3.5 Hz). Rapid [Ca2+]i oscillations were also detected in the acrosomal and postacrosomal regions, as well as in the distal midpiece. Oscillations were not eliminated by dampening the flagellar bending with methyl cellulose. The [Ca2+]i oscillations detected in sperm are significantly more rapid than oscillations detected in other cell types.