To study the effect of rosiglitazone (RSG) on insulin secretion in isolated pancreatic islets under chronic exposure to free fatty acid (FFA) and to investigate the potential signaling mechanism of RSG action.Rat pancreatic islets were cultured with or without FFA (2 mmol/L, oleate:palmitate, 2:1) in the presence or absence of RSG (0.05-10 micromol/L). The insulin release was measured by radioimmuoassay, the expression level of insulin receptor substrate-2 (IRS-2) protein and the association of IRS-2 with p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were determined by immunoprecipitation and Western blot.The islets exposed to high FFA concentration showed an increased basal and a decreased glucose-induced insulin release as compared with control islets (P<0.01). IRS-2 protein level was decreased by 65 % (P<0.01) and the association of IRS-2 with p85 subunit of PI 3-kinase and was decreased by 73 % (P<0.01). When islets were cultured with FFA in the presence of RSG 5 micromol/L, both basal and glucose-induced insulin secretion were reversed to a pattern of control islets (P<0.01, P<0.05). The addition of RSG in the cultured medium increased significantly the expression of IRS-2 protein by 2.6 fold (P<0.01) and the association of IRS-2 with p85 by 2.7-fold (P<0.01) as compared with islets incubated with FFA alone. The effects of RSG on insulin secretion were blocked by a PI 3-kinase inhibitor, wortmannin.The effects of RSG on insulin secretion could be mediated through an IRS-2-associated PI 3-kinase signaling pathway.