Abstract. TSH is a trophic factor for cultured rat thyroid cells (FRTL-5). In the present study we have investigated the mechanism by which TSH promotes cell growth and evaluated the possible role of the adenylate cyclase (AC)-cAMP system in this process. The mitogenic activity of several agents was evaluated by measuring their effect on cell number or 3H-thymidine incorporation into DNA. Forskolin and cholera toxin, two potent and specific activators of the AC, induced a dose dependent increase of 3H-thymidine incorporation. The maximal stimulation, observed at concentrations of 10 μm and 10 ng/ml, respectively, was β 80% of that obtained with optimal concentrations of TSH. A similar effect was obtained with a Graves' IgG preparation (0.2 mg/ml) able to stimulate the thyroid AC or with 3-isobutyl-l-methyl-xanthine (IBMX, 0.5 mm), a phosphodiesterase inhibitor. 8-bromo cAMP (0.5 mm), a cAMP analog, also stimulated 3H-thymidine incorporation, and its potency was ∼ 60% of that of TSH. Similar results were obtained when the mitogenic activity of these compounds was evaluated by cell number. Norepinephrine (NE, 10 μm), although devoid of AC stimulatory activity in these cells, also stimulated 3H-thymidine incorporation, but its potency was only 20–30% of that of TSH. Indomethacin (100 μm), an inhibitor of phospholipid and arachidonic acid metabolism, was able to inhibit the stimulatory effect of NE (84%), and to a lesser extent of TSH (63%) and cholera toxin, had minor effect on forskolin (24%), IBMX (16%) and Graves' IgG (8%), and no effect on 8-bromo cAMP. The results of the present study indicate that: 1) 8-bromo cAMP, as well as agents which stimulate the thyroid adenylate AC (cholera toxin, forskolin or Graves' IgG) or inhibit intracellular cAMP degradation (IBMX) promote FRTL-5 cell proliferation; 2) NE, although devoid of AC stimulatory activity in FRTL-5 cells, also stimulates 3H-thymidine incorporation; 3) indomethacin has a different inhibitory effect on stimulated 3H-thymidine incorporation. These data suggest that cAMP is involved in FRTL-5 cell proliferation, but other biochemical events, independent of the AC-cAMP system and indomethacin sensitive, are operating and may be required to achieve the full activation of the replicative machinery, as obtained with TSH.