Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on the mucosal mast cell line, RBL-2H3, results in the rapid and persistent tyrosine phosphorylation of Vav. Immunoprecipitation of Vav from activated cells revealed co-immunoprecipitated phosphoproteins of molecular weights identical to the Fc epsilon RI beta and gamma chains, and the former was reactive with antibody to the Fc epsilon RI beta chain. Conversely, Western blots revealed the presence of p95 Vav in Fc epsilon RI immunoprecipitates. The association of Vav and of Grb2 with the receptor was found to be regulated by aggregation of the receptor, and the interaction of Vav with the Fc epsilon RI was localized to the gamma chain. To gain insight on the signaling pathway in which Vav participates, we investigated the in vivo associations of Vav with other molecules. A reducible chemical cross-linking agent was used to covalently maintain protein interactions under nonreducing conditions. A fraction of Vav increased in mass to form a complex of >300 kDa in molecular mass. Under reducing conditions the cross-linked Vav immunoprecipitates showed the presence of Grb2, Raf-1, and p42 super(mapk) (ERK2). In vitro kinase assays of Raf-1 activity associated with Vav revealed that this complex had an activity greater than that of Raf-1 derived from nonactivated cells, and aggregation of the Fc epsilon RI did not modulate this activity. In contrast, aggregation of the Fc epsilon RI increased the total Raf-1 activity by 2-5-fold. These results demonstrate that Vav associates constitutively with components of the mitogen-activated protein kinase pathway to form an active multimeric signaling complex whose in vivo activity and associations may be directed by aggregation of the Fc epsilon RI. The findings of this study may also be relevant to other members of the immune recognition receptor family that share the T-cell antigen receptor zeta / gamma chains.