This study was conducted to develop an isolation method for adult pig islets and to investigate in vivo function after xenotransplantation as well as in vitro function of isolated islets. From the splenic portion of the glands (n = 20), 3277 +/- 645 islets per gram of pancreas were isolated and recovered by a five-step dextran discontinuous density gradient method. Purity of the final preparation obtained from 20 different consecutive pig pancreata was 80-90%. In vitro incubation study revealed a significant insulin release from isolated adult pig islets in response to glucose stimulation. In vitro perifusion study demonstrated a biphasic insulin response to glucose stimulation from isolated islets. Xenotransplantation of approx 2000 isolated adult pig islets into the portal vein of diabetic Wistar rats (n = 8) significantly reduced serum glucose levels from 431 +/- 24 mg/dL to 173 +/- 18 mg/dL 24 h after transplantation. This study provides a development of an isolation method for adult pig islets and demonstrates that isolated islets are viable and functioning both in vitro study and in vivo study of xenotransplantation.