Calcium plays a role in sperm motility hyperactivation and the acrosome reaction, but the relationship between cytoplasmic calcium (Ca2+in) levels in the two states was heretofore unknown. The Ca2+ indicator indo-1 was used to detect Ca2+in in moving hamster sperm in two sets of experiments. In the first experiment, activated, hyperactivated, and zona pellucida-induced acrosome-reacted/hyperactivated sperm were analyzed at the time of peak of activity for each state. In the second experiment, sperm in all states were analyzed at one time point. In both sets, mean Ca2+in in the acrosomal region, postacrosomal region, and flagellar midpiece was greater in hyperactivated sperm than in activated sperm, and in acrosome-reacted/hyperactivated sperm than in unreacted/hyperactivated sperm (P < 0.001). Ca2+in had increased to a greater extent in the midpiece than in the head in hyperactivated sperm, while the reverse was true for acrosome-reacted sperm. Oscillations at the frequency of the flagellar beat cycle were detected chiefly in the proximal flagellar midpiece of acrosome-reacted sperm, as they had been previously reported to occur in activated and hyperactivated sperm. Thus, Ca2+in may be maintained at two different elevated levels in sperm, and continues to oscillate after the acrosome reaction.