To determine the role of the myristoylated amino terminus of Galpha in G protein activation, nine individual cysteine mutations along the myristoylated amino terminus of Galpha(i) were expressed in a functionally Cys-less background. Thiol reactive EPR and fluorescent probes were attached to each site as local reporters of mobility and conformational changes upon activation of Galpha(i)GDP by AlF(4)(-), as well as binding to Gbetagamma. EPR and steady state fluorescence anisotropy are consistent with a high degree of immobility for labeled residues in solution all along the amino terminus of myristoylated Galpha(i). This is in contrast to the high mobility of this region in nonmyristoylated Galpha(i) [Medkova, M., et al. (2002) Biochemistry 41, 9962-9972]. Steady state fluorescence measurements revealed pronounced increases in fluorescence upon activation for residues 14-17 and 21 located midway through the 30-amino acid stretch comprising the amino-terminal region. Collectively, the data suggest that myristoylation is an important structural determinant of the amino terminus of Galpha(i) proteins.