Guanylyl cyclase C (GC-C) is the receptor for the heat-stable enterotoxin produced by bacteria as well as for the newly discovered mammalian hormones guanylin and uroguanylin. Ligand activation of GC-C causes it to produce cyclic GMP inside target cells. Although once thought to be restricted to the intestine, GC-C mRNA has recently been detected in other tissues. We now examine the expression, localization, and activation of this glycoprotein after partial hepatectomy in rats. By immunoblot analysis, GC-C protein appeared as early as 4 h after partial hepatectomy, reached its maximal expression (a 30-fold increase) between 24 and 48 h, and returned to low baseline levels at 96 h. During the regenerative period, we detected two GC-C isoforms that differed in their size, temporal expression, and carbohydrase sensitivities. We showed that 131- and 140-kDa GC-C isoforms represented immature and mature GC-C glycoforms on the basis of endoglycosidase H and PNGase sensitivities. Cell separation experiments revealed that the nonparenchymal cell fractions of regenerating liver contained four times as much GC-C as purified hepatocytes. Immunohistochemistry confirmed these findings. The exuberant expression of GC-C by nonparenchymal cells and, to a lesser extent, hepatocytes suggests a role for cyclic GMP in liver regeneration.