The adenylate cyclase stimulator forskolin increases intracellular cyclic AMP (cAMP) in rat FRTL-5 cells within minutes and, after a lag phase of 20-24 h, an increase of cells in metaphase is seen. The dose-response relationships were similar in both systems, with significant increases in the number of metaphases observed at approximately 0.1 mumol/l and a doubling of cAMP levels at 1 mumol/l, whilst doses of 0.1 mmol/l and above proved cytotoxic. An involvement of intracellular cAMP as a positive intermediate in cell division was further suggested by the finding that a low dose of forskolin (0.1 mumol/l) potentiated TSH stimulation of mitosis. Isobutyl methyl xanthine (IBMX), a phosphodiesterase inhibitor, also acted as a mitogen and potentiated TSH action. Moreover, the simultaneous inclusion of low doses of IBMX and forskolin additionally potentiated TSH stimulation of mitosis. An analogue of cAMP, dibutyryl cAMP, also stimulated mitosis and acted over a restricted dose range, with maximal stimulation at 1 mmol/l. We conclude that cAMP may act as a positive signal for FRTL-5 thyroid cell proliferation.