Vav has structural features found in signaling proteins and is expressed only in hematopoietic cells. The recent development of mice Vav -/- has confirmed a major role of Vav in early blood cell development. We previously showed that Vav constitutively interacts with glutathione-S-transferase-Grb2. Coimmunoprecipitation experiments supported the idea of a complex formed by Vav-Grb2 in vivo. This complex is of potential interest in signaling of hematopoietic cells. In this work we localize the domains of Vav and Grb2 involved in this interaction. By the use of an in vivo genetic approach (the double hybrid system) and through in vitro experiments (glutathione-S-transferase fusion proteins) we furnish evidence that the interaction between Vav and Grb2 involves the C-SH3 domain of Grb2 and the proline-rich region located in the N-SH3 of Vav. Furthermore this was confirmed by the use of both Vav and Sos derived proline-rich peptides which blocked the binding. In addition we show that Vav also interacts with Grb3-3, a naturally occurring Grb2 isoform wich lacks functional SH2 domain.