Fas/APO-1(CD-95) activation induced rapid apoptotic cell death of primary rat hepatocytes in suspension culture. Activators of cAMP-dependent protein kinase (glucagon and N6-benzoyl-cAMP) protected against apoptosis, whereas the specific cAMP-kinase inhibitor (Rp)-8-Br-cAMPS enhanced Fas-induced death. The latter observation indicated that even the basal cAMP level may provide partial protection against Fas-induced hepatocyte apoptosis. Two-dimensional gel electrophoresis revealed decreased phosphorylation of several proteins in Fas-activated cells. Most of these dephosphorylations were attenuated or not observed in cells simultaneously stimulated by anti-Fas and cAMP, indicating a tight correlation between the dephosphorylations and death. Elevation of cAMP rescued the cells not only from the Fas-induced morphological changes and dephosphorylation, but also from functional deterioration. Whereas cells treated with anti-Fas alone quickly lost plating efficiency, hepatocytes co-treated with glucagon retained their ability to adhere and spread on a collagen substratum.