Fertilization of the mammalian oocyte depends on the ability of spermatozoa to undergo a process known as capacitation as they ascend the female reproductive tract. A fundamental feature of this process is a marked increase in tyrosine phosphorylation by an unusual protein kinase A (PKA)-mediated pathway. To date, the identity of the intermediate PKA-activated tyrosine kinase driving capacitation is still unresolved. In this study, we have identified SRC as a candidate intermediate kinase centrally involved in the control of sperm capacitation. Consistent with this conclusion, the SRC kinase inhibitor SU6656 was shown to suppress both tyrosine phosphorylation and hyperactivation in murine spermatozoa. Moreover, SRC co-immunoprecipitated with PKA and this interaction was found to lead to an activating phosphorylation of SRC at position Y416. We have also used difference-in-2D-gel-electrophoresis (DIGE) in combination with mass spectrometry to identify a number of SRC substrates that become phosphorylated during capacitation including enolase, HSP90 and tubulin. Our data further suggest that the activation of SRC during capacitation is negatively controlled by C-terminal SRC kinase. The latter was localized to the acrosome and flagellum of murine spermatozoa by immunocytochemistry, whereas capacitation was associated with an inactivating serine phosphosphorylation of this inhibitory kinase.