Previous studies have shown the efficacy of betacellulin (BTC) to promote P-cell regeneration. Because of its short half-life, however, the effect of BTC may have been underestimated. This study was conducted to assess the effect of continuous administration of BTC on P-cell regeneration. Adenovirus vectors encoding proBTC (Ad-proBTC) and mature BTC (Ad-mBTC) were prepared, and the efficacy of secretion of BTC was compared in AML12 hepatocytes. When AML12 cells were infected with Ad-proBTC or Ad-mBTC, cells infected with Ad-mBTC secreted considerably larger amount of BTC. We then infused Ad-mBTC into the mouse tail vein. Expression of BTC was detected in the liver for at least 21 days, and serum BTC was maintained at approximately 1 ng/ml for 7 days. When Ad-mBTC was infused immediately after administration of STZ (170 mg/kg), elevation of the plasma glucose induced by STZ was markedly inhibited, and the plasma glucose concentration remained at less than 200 mg/dl for 21 days. The insulin content and the P-cell mass were significantly increased in Ad-mBTC-infused mice. These results indicate that continuous administration of BTC is quite effective in promoting regeneration of P-cells.