We have previously shown that cAMP protects against hydrophobic bile acid-induced apoptosis in cultured rat hepatocytes through pathways dependent on activation of phosphoinositide 3-kinase and inhibition of mitogen activated protein kinase. Hepatocyte growth factor protects epithelial cells against apoptosis and activates both of these kinases in hepatocytes. We studied the effect of hepatocyte growth factor on glycochenodeoxycholate-induced apoptosis to determine whether hepatocyte growth factor protects hepatocytes against bile acid-induced apoptosis and whether the protective effect is mediated via phosphoinositide 3-kinase and/or mitogen-activated protein kinase pathways. Two-hour exposure of cultured rat hepatocytes to glycochenodeoxycholate resulted in apoptosis in 12.5 ± 0.49% of the cells. Pretreatment with hepatocyte growth factor (50 ng/mL) decreased apoptosis by 50% to 70%. Hepatocyte growth factor cytoprotection was prevented by pretreatment with the phosphoinositide 3-kinase inhibitors, wortmannin (50 nmol/L) or Ly 294002 (40 μmol/L). Hepatocyte growth factor activated phosphoinositide 3-kinase dependent protein kinase B and mitogen-activated protein kinase. Pretreatment of hepatocytes with a mitogen-activated protein kinase inhibitor, U0126 (40 μmol/L) or an inhibitor of pp70s6 kinase, rapamycin (100 nmol/L), had no effect on the growth factor's anti-apopotic effect. Treatment with hepatocyte growth factor resulted in mitogen-activated protein kinase-dependent phosphorylation of BAD on serine112. In summary, hepatocyte growth factor protection against bile acid-induced apoptosis occurs via a phosphoinositide 3-kinase pathway and is not dependent on the mitogen-activated protein kinase pathway, phosphorylation of BAD on serine112, or activation of p70S6 kinase.