Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid–induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5% ± 1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 μmol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2% ± 3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 μmol/L), glucagon (200 nmol/L), and a combination of forskolin (20 μmol/L) and 3-isobutyl-1-methylxanthine (20 μmol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP–induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 μmol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. In conclusion: 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid–induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid–induced apoptosis appears to involve PKA, MAPK, and PI3K.