Galanin, an inhibitor of insulin secretion in pancreatic beta-cells, exerts its multiple effects through mechanisms that are sensitive to pertussis toxin (PTX). G proteins have been characterized in RINm5F cells. By ADP ribosylation and immunoblotting, the alpha-subunits of Gi1, Gi2, Gi3, and two forms of Go were identified, Gi alpha 2 being predominant. As expected from a G protein-linked receptor, GTP and its nonhydrolyzable analogue GTP-gamma-S decreased tracer galanin binding to cell membranes. This resulted from a change in receptor affinity without any modification in the number of sites. Selective antibodies against the COOH-terminal decapeptide of the alpha-subunits of the Gi and Go proteins were used to block G protein interaction before we studied galanin binding. Antibody AS, which selectively recognizes Gi alpha 1 and Gi alpha 2, decreased tracer galanin binding to membranes at concentrations where there were no effects of other antibodies specifically directed against Gi alpha 3 or G alpha o. These data suggest that Gi1 and/or Gi2 interact with the galanin receptor and probably mediate the effects of galanin in pancreatic beta-cells.