Previous pharmacologic and genetic studies have demonstrated a critical role for the low molecular weight GTP-binding protein RhoA in the regulation of cell-mediated killing by cytotoxic lymphocytes. However, a specific Rho family guanine nucleotide exchange factor (GEF) that activates this critical regulator of cellular cytotoxicity has not been identified. In this study, we provide evidence that the Rho family GEF, Vav-2, is present in cytotoxic lymphocytes, and becomes tyrosine phosphorylated after the cross-linking of activating receptors on cytotoxic lymphocytes and during the generation of cell-mediated killing. In addition, we show that overexpression of Vav-2 in cytotoxic lymphocytes enhances cellular cytotoxicity, and this enhancement requires a functional Dbl homology and Src homology 2 domain. Interestingly, the pleckstrin homology domain of Vav-2 was found to be required for enhancement of killing through some, but not all activating receptors on cytotoxic lymphocytes. Lastly, although Vav and Vav-2 share significant structural homology, only Vav is able to enhance nuclear factor of activated T cells-activator protein 1-mediated gene transcription downstream of the T cell receptor. These data demonstrate that Vav-2, a Rho family GEF, differs from Vav in the control of certain lymphocyte functions and participates in the control of cell-mediated killing by cytotoxic lymphocytes.