The requirement for external Ca+' during capacitation of ejaculated bovine sperm with heparin and changes in sperm-associated 45Ca +' during capacitation were investigated in vitro. Sperm capacitation was evaluated by ability to undergo an acrosome reaction (AR) upon exposure to lysophosphatidylcholine. The percentage of sperm which were capacitated during a 4 h incubation with heparin increased exponentially with increased exposure time to 2 mM Ca+'. When sperm were incubated with or without heparin in the presence of 45CaC12, there was no difference in the amount of 45Ca+2 associated with sperm initially or at 1 h of incubation. Incubation with heparin resulted in a greater amount of sperm-associated 45Ca+' at 2, 3, and 4 h as compared to sperm incubated without heparin. The amount of 45Ca+2 associated with sperm during capacitation was unaffected by washing with 2 mM EGTA-5 mM LaC1,. Glucose (5 mM) inhibited the effects of heparin on sperm-associated 45Ca+2 and on capacitation. The inhibitory effects of glucose could be overridden by 8-bromo-CAMP. The results suggest that the requirement for external Ca+' during capacitation with heparin may be related to an increased association of external Ca+' with sperm.