Rap1b has been implicated in the transduction of the cAMP mitogenic signal. Rap1b is phosphorylated and activated by cAMP, and its expression in cells where cAMP is mitogenic leads to an increase in G(1)/S phase entry and tumor formation. The PCCL3 thyroid follicular cells represent a differentiated and physiologically relevant system that requires thyrotropin (TSH), acting via cAMP, for a full mitogenic response. In this model system, cAMP stimulation of DNA synthesis requires activation and phosphorylation of Rap1b by the cAMP-dependent protein kinase A (PKA). This scenario presents the challenge of identifying biochemical processes involved in the phosphorylation-dependent Rap1b mitogenic action. In thyroid cells, Akt has been implicated in the stimulation of cell proliferation by TSH and cAMP. However, the mechanism(s) by which cAMP regulates Akt activity remains unclear. In this study we show that in PCCL3 cells 1) TSH inhibits Akt activity via cAMP and PKA; 2) Rap1b is required for cAMP inhibition of Akt; and 3) transduction of the cAMP signal into Akt requires activation as well as phosphorylation of Rap1b by PKA.