Progesterone at 3 μM triggers a biphasic (transient and sustained) increase in intracellular calcium ([Ca²⁺]_i) in human sperm, which is believed to be a prerequisite for progesterone-induced acrosome reaction (AR). As very little is known about how AR occurrence, latency, and completion relate to the characteristics of the progesterone-induced [Ca²⁺]_i signal, we examined these events using fluorescence microscopy of individual living human sperm. Direct assessment of acrosomal status after calcium imaging showed no differences in kinetics or amplitude of the preceding progesterone-induced calcium responses in acrosome-reacted and acrosome-intact cells, which indicates that the amplitude of the [Ca²⁺]_i signal is not the critical determinant of AR. Chelation of extracellular calcium to arrest AR at varying times after progesterone stimulation revealed that maximal AR occurred immediately following progesterone stimulation, during the initial transient calcium influx rather than during the sustained calcium response. Attempts to follow acrosomal dispersal in real-time by staining with the acidic organelle probes LysoTracker DND-99 and dapoxyl (2-aminoethyl) sulphonamide (DAES) proved inconclusive due to heterogeneous labeling of the cell population. Surprisingly, the dye was often not confined to the acrosome but stained the whole sperm head, which suggests that only a subpopulation of human sperm cells contains a sufficiently acidic acrosome.