The hypothesis that intracellular [Ca2+] is a cell parameter responsive to extreme temperatures in rat meiotic and postmeiotic spermatogenic cells was tested using intracellular fluorescent probes for Ca2+ and pH. In agreement with this hypothesis, extreme temperatures induced a rapid increase of cytosolic [Ca2+] in rat pachytene spermatocytes and round spermatids. Oscillatory changes in temperature can induce oscillations in cytosolic [Ca2+] in these cells. Intracellular [Ca2+] homeostasis in round spermatids was more sensitive to high temperatures compared with pachytene spermatocytes. The calculated activation energies for SERCA ATPase-mediated fluxes in pachytene spermatocytes and round spermatids were 62 and 75 kJ mol-1, respectively. The activation energies for leak fluxes from intracellular Ca2+ stores were 55 and 68 kJ mol-1 for pachytene spermatocytes and round spermatids, respectively. Together with changes in cytosolic [Ca2+], round spermatids undergo a decrease in pHi at high temperatures. This temperature-induced decrease in pHi appears to be partially responsible for the increase in cytosolic [Ca2+] of round spermatids induced by high temperatures. This characteristic of rat meiotic and postmeiotic spermatogenic cells to undergo an increment in cytosolic Ca2+ at temperatures > 33°C can be related to the induction of programmed cell death by high temperatures in these cells.