Thymosin fraction five (TF5), a well-characterized immunoregulatory thymic preparation, has been reported to stimulate corticotropin (ACTH) release from rat pituitary cells. Since a previous study in our laboratory had shown that TF5 was able to stimulate ACTH release from corticotropin-releasing hormone (CRH)-insensitive corticotropic tumor cells, it was of interest to assess the role of calcium in the mechanism of action of TF5 on corticotropic cells. A CRH-insensitive variant, denoted AtT-20(CI), of the wild-type corticotropic tumor cell line AtT-20 was used. Synthetic h/rCRH within a dose range of 0.1-100 nM was completely ineffective to stimulate basal ACTH release from AtT-20(CI) cells, although the same batch of neuropeptide displayed the expected ACTH-releasing activity on dispersed rat pituitary cells (for instance, 0.1 nM CRH induced a 3.7-fold increase in ACTH release in this cell system). Median eminence extracts (1/10) induced only a 12% increase in ACTH release from AtT-20(CI) cells as compared to the 395% stimulation induced in normal pituitary cells. As expected, TF5 induced a dose-dependent increase in ACTH release from AtT-20(CI) cells. However, this ACTH-releasing activity of TF5 was completely abolished when cells were incubated in Ca-free medium or Ca-free medium containing 0.5 mM EGTA. On the other hand, the presence of the Ca ionophore A23187 (5 µM) in medium containing normal Ca levels (2.5 mM) did not affect the ACTH-releasing activity of TF5 on AtT-20(CI) cells. Our results suggest that thymosin peptides exert their ACTH-releasing effect on corticotrophs by a Ca-dependent mechanism which does not require the integrity of the CRH receptor-associated activation pathway.