Rises in intracellular Ca² concentration ([Ca² ]_i) caused by progesterone, an inducer of the acrosome reaction, or by cyclic nucleotides, possible second messengers, were investigated by Ca² imaging of the head of individual mouse sperm. Progesterone induced a [Ca² ]_i rise in a dose-dependent manner (4–40 μM), primarily in the postacrosomal region. For 20-μM progesterone, Ca² responses occurred in 42% of sperm, separated into two types: transient type (60% of responding cells; duration, 1–1.5 min; mean amplitude, 335 nM) and prolonged type (40%; >3 min; 730 nM). Prolonged responses required higher doses of progesterone, and their occurrence was enhanced significantly by preincubation for 2–4 h as compared with transient responses. 8-Bromo-cGMP (0.3–3 mM) induced a [Ca² ]_i rise more effectively than did 8-bromo-cAMP. For 1-mM 8-bromo-cGMP, 90% of cells exhibited transient Ca² responses (∼1 min; 220 nM), independently of the preincubation time. In Ca² -free medium, most sperm showed no Ca² response to progesterone and 8-bromo-cGMP. Pimozide, a Ca² channel blocker, completely blocked prolonged responses and partially inhibited transient responses. These results suggest that progesterone activates at least two distinct Ca² influx pathways, with fast or slow inactivation kinetics, and some sperm show both types of response. A cyclic nucleotide-mediated process could participate in the progesterone-induced [Ca² ]_i rise.