Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Gαi1. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Gαi1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here, we demonstrate that the R6A-1 peptide interacts with Gα subunits representing all four G protein classes, acting as a core motif for Gα interaction. This contrasts with the consensus G protein regulatory (GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Gαi/o-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Gαi1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Gα subunits and excludes association with Gβγ. R6A-Gαi1 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 Gαi1/Gαs chimeras identify two regions of Gαi1 (residues 1-35 and 57-88) as determinants for strong R6A-Giα1 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.