Activin A and betacellulin (BTC) are thought to regulate differentiation of pancreatic beta-cells during development and regeneration of beta-cells in adults. In the present study, we used neonatal rats treated with streptozotocin (STZ) to investigate the effects of activin A and BTC on regeneration of pancreatic beta-cells. One-dayold Sprague-Dawley rats were injected with STZ (85 mug/g) and then administered for 7 days with activin A and/or BTC. Treatment with activin A and BTC significantly reduced the plasma glucose concentration and the plasma glucose response to intraperitoneal glucose loading. The pancreatic insulin content and beta-cell mass in rats treated with activin A and BTC were significantly increased compared with the control group on day 8 and at 2 months. Treatment with activin A and BTC significantly increased the DNA synthesis in preexisting beta-cells, ductal cells, and 8-cells. The number of islet cell-like clusters (ICCs) and islets was significantly increased by treatment with activin A and BTC. In addition, the number of insulin/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells was significantly increased. These results indicate that, in neonatal STZ-treated rats, a combination of activin A and BTC promoted regeneration of pancreatic beta-cells and improved glucose metabollism. in adults.