It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC ζ activation and insulin-induced tyrosine phosphorylation of Giα to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01∼1 ng/ml PTX for 2 hours, followed by stimulation with 10–100 nM insulin or 1 μM tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [ 3H]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [ 3H]2-DOG uptake, without affecting basal [ 3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC ζ translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [ 125I]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa α-subunit of G-protein (Giα-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Giα-2 antibody. Insulin-induced tyrosine phosphorylation of Giα-2 was found by immunoblot analysis with anti-Giα-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Giα-2 with GTP and PI 3-kinase-PKC ζ signalling by releasing of Gβγ via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues.