We studied the role of various intracellular pathways in thyroglobulin secretion. The P2 agonists (ATP, ADP, GTP), 12-O-tetradecanoylphorbol-13-acetate (TPA), and protein kinase A activators stimulate thyroglobulin secretion in cells grown without TSH. The effects of these agents are additive. Pertussis toxin partially inhibits the effect of ATP but has no effect on the action of GTP. ATP and GTP increase cytosolic calcium (279 +/- 16% and 302 +/- 22%, respectively) while TPA and TSH (1 mU/ml) do not. Thus, both the protein kinase A and kinase C pathways regulate thyroglobulin secretion in FRTL-5 cells.