We have tested the putative role of G-protein β-subunits in insulin exocytosis by transient expression of βγ-binding proteins targeted to the plasma membrane. The PH domain of the G-protein-linked receptor kinase 2 fused to the transmembrane domain of a cell surface receptor and the α-subunit of the retinal G-protein transducin inhibited stimulated insulin release from intact and permeabilised HIT-T15 cells. This effect cannot be imputed to an increase in free Gα, as the RGS protein RGS3 did not reverse this effect. Among the isoforms of Gβ examined, Gβ2 was detected on the plasma membrane by confocal immunomicroscopy. These observations suggest a role for G-protein βγ-subunits in insulin exocytosis.