We previously identified a novel murine protein, AND-34, with a carboxyl-terminal domain homologous to Ras family guanine nucleotide exchange factors (GEFs), which bound to the focal adhesion docking protein p130Cas. Work by others has implicated both the human homologue of AND-34, BCAR3, and human p130Cas, BCAR1, in the resistance of breast cancer cells to the anti-estrogen tamoxifen. Here we report that AND-34 displays GEF activity on RalA, Rap1A, and R-Ras but not Ha-Ras GTPases in cells. In contrast to several other Ral-GEFs, the Ral GEF activity of AND-34 is not augmented by constitutively active Ha-RasVal-12, consistent with the absence of a detectable Ras-binding domain. Efficient binding to AND-34 required both the Src-binding domain and a flanking carboxyl-terminal region of p130Cas. The p130Cas-binding site mapped to a carboxyl-terminal sequence within the AND-34 GEF domain. Overexpression of p130Cas, but not an AND-34-binding mutant of p130Cas, inhibited the Ral GEF activity of co-transfected AND-34. This work identifies a new potential function for p130Casand a new regulatory pathway involved in the control of Ral, Rap, and R-Ras GTPases that may participate in the progression of breast cancer cells to tamoxifen resistance.