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G proteins may serve as targets for pharmacological activation of signaling pathways bypassing the regulatory events that may counteract the effect of the external stimulus on the level of receptors. We sought to identify peptides as lead structures interacting with G proteins utilizing a commercially available phage-display library. The heptapeptide library was screened for binding to human Gαi1 which was modified with a hexahistidine tag and immobilized on a Ni2+-coated surface. After several rounds of phage selection a number of phage clones with consensus sequences were found. Peptides with such sequences were chemically synthesized and their effect on [35S]GTPγS binding to Gαi1 and other G protein α subunits was determined. Through this process two peptide ‘families’ with the capability to activate G proteins were identified. The peptides showed no structural similarity to known peptide or nonpeptide G protein activators with a cationic amphiphilic structure like mastoparan or alkylamines. The functional relevance of the peptide-G protein interaction was shown by an increased sensitivity for guanine nucleotides of high-affinity agonist binding to A1 adenosine receptors. The peptide G protein activators may, therefore, serve as novel tools for further investigation of receptor-independent activation of G proteins.