The function of Gly-60, the conserved glycine in the DXXG domain of v-H-ras, was examined by site-directed mutagenesis. It was found that while the G60A (Gly-60 to Ala substitution) mutation has little effect on the interaction of H-ras with guanine nucleotides, it completely abolishes the biological activity of v-H-ras. The G60A mutation also exerts little effect on the interaction of H-ras with SDC25C (a guanine nucleotide exchange factor) and GAP. However, the G60A mutation does lower the ability of H-ras to bind Raf. GTP induces an enhancement of fluorescence emission in complexes consisting of H-ras and the fluorescent dye 8-anilino-1-naphthalenesulfonic acid. This enhancement is blocked by the G60A mutation. On the basis of these observations, we propose that the GTP-induced conformational change of H-ras, a process required for H-ras activities, is impaired by the G60A mutation.