Flow cytometry was utilized to measure the influx of calcium into fresh and frozen-thawed equine and bovine spermatozoa that had been treated with either dilauroyl-phosphatidylcholine ( PC-12) or the calcium ionophore A23187 (A23187) to initiate sperm capacitation. Sperm suspensions were stained with the fluorescent calcium indicator, Fluo-3, to measure intracellular calcium concentrations and with propidium iodide (PI) to evaluate sperm viability. After staining, sperm were incubated at 37o C in a Hepes buffered salt medium (BGM3,control) with either PC-12 or A23187 and analyzed just prior to (time 0) and 1, 5, 15 and 30 min after addition of capacitating agents. When fresh stallion sperm were treated with either BGM3 (control) or PC-12 neither the percentages of viable sperm or the percentages of sperm with increased levels of intracellular calcium, changed during the incubation period. However, when fresh stallion sperm were treated with A23187, within 15 min of incubation, the percentages of sperm exhibiting high levels of intracellular calcium increased. Treatment of frozen-thawed stallion sperm with A23187 induced an immediate increase in intracellular calcium, while the percentages of control and PC-12 treated sperm having elevated intracellular calcium levels increased after 5 min of incubation. A23187 induced an immediate calcium uptake into both fresh and frozen-thawed bull sperm, while control sperm and sperm treated with PC-12 increased intracellular Ca++ levels only after 15 min of incubation. These results indicate that cryopreservation alters the plasma membranes sperm permitting a rapid influx of calcium into the sperm, which was not observed in fresh sperm. In addition, calcium ionophore A23187 induces a rapid influx of calcium into both stallion and bull spermatozoa