The endpoint of in vitro/in vivo capacitation is the ability of sperm surface receptors to bind to their complementary ligands on zona pellucida, the extracellular glycocalyx that surrounds the egg, and undergo the Ca2+-dependent signal transduction. The net result is the fenestration and fusion of the sperm plasma membrane and the underlying outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents. The hydrolytic action of glycohydrolases and proteinases, released at the site of sperm-zona binding, along with the enhanced thrust generated by the hyperactivated flagellar motility of the bound spermatozoon, are important factors that regulate the fertilization process. This report discusses the physiological significance of calmodulin, a 17 kDa Ca2+ sensor protein, in sperm function. The in vitro experimental approaches described in this article provide evidence strongly suggesting that calmodulin plays an important role in the priming (that is, capacitation) of mouse spermatozoa as well as in the agonist-induced acrosome reaction. In addition, we have used several calmodulin antagonists in an attempt to characterize further the morphological and biochemical changes associated with sperm capacitation. Data presented in this report suggest that calmodulin antagonists prevent capacitation by interfering with multiple regulatory pathways and do so either with or without adverse effects on sperm motility and protein tyrosine phosphorylation of sperm components.