Human cells contain four homologous Ras proteins, but it is unknown whether these homologues have different biological functions. As a first step in determining if Ras homologues might participate in distinct signaling cascades, we assessed whether a given Ras guanine nucleotide exchange factor could selectively activate a single Ras homologue in vivo. We found that Ras-GRF/Cdc25Mm activates Ha-Ras, but does not activate N-Ras or K-Ras 4B, protein in vivo. Moreover, our results suggested that residues within the C-terminal hypervariable domains of Ras proteins may dictate, at least in part, the specificity of Ras-GRF/CDC25Mm for Ha-Ras protein. Our studies represent the first biochemical evidence that a Ras GEF can selectively activate a single Ras homologue in vivo. Selective activation of a single Ras homologue by Ras-GRF/Cdc25Mm or other Ras guanine nucleotide exchange factors could potentially enable each of the Ras homologues to participate in different signal transduction pathways.