Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) 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.