Increase of intracellular cyclic adenosine monophosphate by the permeant cyclic adenosine monophosphate analog, 8-(4-chlorophenylthio)-adenosine 3 H :5 H -cyclic monophosphate, is mitogenic for normal adult rat chromafยฎn cells. The mitogenic effect is blocked by the phosphatidylinositol 3-kinase inhibitor, LY294002, and is associated with accumulation of phosphorylated Akt and p70S6 kinase, suggesting that cyclic adenosine monophosphate activates Type l phosphatidylinositol 3-kinase. The mechanism of activation was examined in PC12 pheochromocytoma cells, which are neoplastic chromafยฎn cells that exhibit many of the biochemical characteristics of their normal counterparts. Incubation of PC12 cells with 8-(4-chlorophenylthio)-adenosine 3 H :5 H -cyclic monophosphate led to a signiยฎcant increase in total phosphatidylinositol 3-kinase activity that was sensitive to low concentrations of LY294002. The increase was maximal at 1 h and returned to basal levels within six hours. Immunoprecipitation studies showed no increase in phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immune complexes from PC12 cells stimulated by 8-(4-chlorophenylthio)-adenosine 3 H :5 H -cyclic monophosphate, in contrast to cells stimulated by nerve growth factor. Instead, activity was demonstrated in association with p110g and p85. These ยฎndings suggest that cyclic adenosine monophosphate causes activation of Types IA and IB phosphatidylinositol 3-kinase by a novel mechanism in chromafยฎn and pheochromocytoma cells. That activation may contribute to chromafยฎn cell proliferation and to the development and progression of pheochromocytomas.