The diterpene forskolin is a potent (100-fold) stimulator of guinea pig thyroid cAMP accumulation with half-maximal activation occurring at 40 microM. Forskolin stimulation is more rapid than that of TSH, attaining a 5-fold increase within 1 min of exposure. The stimulation is also rapidly reversible. The diterpene does not sensitize thyroid cAMP accumulation to TSH, and the concentration yielding half-maximal response is not altered by the presence of low levels of forskolin. At maximally stimulating concentrations, the effects of TSH and forskolin on cAMP accumulation are additive. Forskolin stimulates thyroid adenylate cyclase approximately 10-fold in membranes from several species with half-maximal effects occurring at 3--9 microM through an action on the maximum velocity and not the Km for ATP. The activation of thyroid membranes is readily reversible. Guanyl nucleotides are not required for stimulation by forskolin, and they do not sensitize to forskolin. Moreover, the drug did not sensitize the membrane adenylate cyclase to guanosine 5'-[beta, gamma-imido]triphosphate or to isoproterenol and was equally effective with either Mg++ or Mn++ as the divalent cation. Forskolin stimulation is additive with that of guanyl nucleotides and F-. The site of action of forskolin in the adenylate cyclase complex is uncertain. Data from Bordetella pertussis, testicular, and S-49 lymphoma mutant cyclases suggest that one of the guanyl nucleotide regulatory proteins may be required to promote the forskolin effect. We conclude that forskolin is a useful activator of thyroid adenylate cyclase both in vitro and in intact tissue, which will be useful in elucidating the coupling process of the adenylate cyclase system and in differentiating cAMP-mediated from other forms of activation of the thyroid.