Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G_i{alpha1}. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G_i{alpha1} and acts as a guanine nucleotide dissociation inhibitor (GDI). Here, we demonstrate that the R6A-1 peptide interacts with G_{alpha} subunits representing all four G protein classes, acting as a core motif for G_{alpha} interaction. This contrasts with the consensus G protein regulatory (GPR) sequence, a 28-mer peptide GDI derived from the GoLoco/GPR motif that shares no homology with R6A-1 and binds only to G_i{alpha1-3} in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G_{alpha} subunits and excludes association with G_{beta}. R6A-G_i{alpha1} complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg²⁺, suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G_i{alpha1}/G_s{alpha} chimeras identify two regions of G_i{alpha1} (residues 1-35 and 57-88) as determinants for strong R6A-G_i{alpha1} interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states.