Mice lacking N-acetylglucosaminyltransferase III (GlcNAc-TIII) exhibit slightly but significantly retarded liver tumor progression after a single injection of 10 micro g/g diethylnitrosamine (DEN) and continued administration of phenobarbital (PB) in drinking water. A key question is whether the absence of GlcNAc-TIII inhibits cell proliferation or induces apoptosis. Because PB aids tumor progression, we tested whether it diminished the difference in tumor progression between Mgat3(+/+) and Mgat3(Delta/Delta) mice. Here, we show that in the absence of PB, control males developed about twice as many liver tumor nodules as males lacking GlcNAc-TIII. Both the size of liver tumors and liver weights were significantly greater in DEN-treated wild-type or heterozygous mice. Apoptosis assays performed monthly after DEN treatment showed no differences between mutant and wild-type. However, there was a marked retardation in liver regeneration after partial (70%) hepatectomy (PH). Wild-type mice incorporated bromodeoxyuridine in approximately 15% of hepatocyte nuclei at 48 h after PH, whereas mice lacking GlcNAc-TIII had only approximately 5% positive nuclei. This was not because of enhanced apoptosis in mutant mice after PH. Expression of the Mgat3 gene remained undetectable in wild-type liver by Northern analysis after tumor induction or after PH. In addition, transgenic overexpression of GlcNAc-TIII in hepatocytes did not enhance tumor progression in Mgat3(Delta/Delta) mice, and there were no differences in tumor progression or liver regeneration after PH between control and transgenic mice overexpressing GlcNAc-TIII in liver. Therefore, the nonhepatic action of GlcNAc-TIII promotes hepatocyte proliferation after PH, as well as the progression of DEN-induced tumors, providing evidence for a functional role of the bisecting GlcNAc on circulating glycoprotein growth factor(s) that stimulate hepatocyte proliferation.