Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin α6β1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of αvβ3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-αv or anti-β3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm αvβ3 integrin. Developmental Dynamics 239:773–783, 2010. © 2010 Wiley-Liss, Inc.