We have previously reported that the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) induces, in the TT cell line of human medullary thyroid carcinoma, decreased cellular proliferation, increased calcitonin secretion, and enhanced calcitonin gene transcription (deBustros, A., Baylin, S. B., Berger, C. L., Roos, B. A., Leong, S. S., and Nelkin, B. D. (1985) J. Biol. Chem. 260, 98-104). The cellular responses evoked by TPA are thought to be mediated by protein kinase C. In the present study, we have investigated whether protein kinase A, another key mediator of extracellular signal transduction, may also alter the differentiation status of the TT cells. We find that the effects of cAMP, an activator of protein kinase A, on cellular growth, calcitonin secretion, and calcitonin gene transcription almost parallel those of TPA. We also show that both TPA and cAMP lead to similar increases of both major mRNA species encoded within the calcitonin gene, calcitonin itself and the neuropeptide calcitonin gene-related peptide. In addition, cAMP increases nuclear calcitonin and calcitonin gene-related peptide mRNA precursors to a greater extent (8-10-fold) than it does the mature cytoplasmic mRNA species (2-4-fold). The effects of TPA and cAMP on the TT cells are additive rather than synergistic. Furthermore, TPA evokes no increase in intracellular cAMP. We thus conclude that TPA and cAMP can trigger, independently, in the TT cells, a similarly programmed set of events resulting in a more differentiated phenotype. These cells provide a model system to explore how these two pathways of signal transduction converge to regulate molecular events such as the transcription of the calcitonin gene.