We have investigated the unique role of the insulin receptor (IR) and the balance of its isoforms A and B in the regulation of apoptosis in simian virus 40 (SV40)-immortalized neonatal hepatocytes. Immortalized hepatocytes lacking (HIR KO) or expressing the entire IR (HIR LoxP), and cells expressing either IRA (HIR RecA) or IRB (HIR RecB) have been generated. IR deficiency in hepatocytes increases sensitivity to the withdrawal of growth factors, because these cells display an increase in reactive oxygen species, a decrease in Bcl-x(L), a rapid accumulation of nuclear Foxo1, and up-regulation of Bim. These events resulted in acceleration of caspase-3 activation, DNA laddering, and cell death. The single expression of either IRA or IRB produced a stronger apoptotic phenotype. In these cells, protein complexes containing IRA or IRB and Fas/Fas-associating protein with death domain activated caspase-8, and, ultimately, caspase-3. In hepatocytes expressing IRA, Bid cleavage and cytochrome C release were increased whereas direct activation of caspase-3 by caspase-8 and a more rapid apoptotic process occurred in hepatocytes expressing IRB. Conversely, coexpression of IRA and IRB in IR-deficient hepatocytes rescued from apoptosis. Our results suggest that balance alteration of IRA and IRB may serve as a ligand-independent apoptotic trigger in hepatocytes, which may regulate liver development.