The effects of synthetic antagonists to mammalian vasoactive intestinal polypeptide (VIP), corticotropin-releasing hormone (CRH), and growth hormone-releasing hormone (GHRH) on in vitro thyrotropin (TSH) and growth hormone (GH) secretion by pituitaries of hatchling slider turtles (Pseudemys scripta elegans) were investigated in hopes of understanding the mechanism(s) of action of the native neuropeptides. Pituitary glands were incubated individually in wells, and the culture medium was changed every 2 hr. After determination of basal secretion rates for one 2 hr period, glands were incubated in medium with or without VIP antagonist (100 or 500 nM) for 2 hr followed by two 2 hr periods with antagonist plus 2.5 nM VIP. VIP alone had no effect on TSH or GH secretion at this dose; however, both TSH and GH secretion were significantly elevated by the high dose of the VIP antagonist plus VIP (2.9-fold). In another experiment, glands were exposed to 2.5 kgiml(500-750 nM) antagonists to VIP, CRH, or GHRH for four 2 hr incubation periods. The GHRH and VIP antagonists stimulated significant increases in TSH secretion above basal release, whereas the CRH antagonist produced a significant stimulation of TSH but not GH release, an observation that is consistent with our earlier report for other CRH-like peptides (Denver and Licht: J. Exp. Zool., 251:306-315, '89a). Maximum stimulation by the antagonists (3.5-to 5-fold) was comparable to maximum stimulation by the native neuropeptides at doses ranging from 25 to 250 nM. These results demonstrate that synthetic peptides that can act as antagonists of neuropeptide actions in mammalian systems have agonistic effects on in vitro TSH and GH secretion by turtle pituitaries. These observations also lend support to the hypothesis that neuropeptide control of the ectotherm pituitary is generally less specific than in mammals.