The ability of thyrotropin-releasing hormone (TRH) to stimulate thyrotropin (TSH) from pituitaries of adult male lizards, Anolis carolinensis, was tested in vivo and in vitro. TSH output by pituitaries in vitro was determined by bioassay of the incubation medium using in vitro T4 output by thyroids from the same lizards. Pituitaries incubated without TRH had no detectable TSH secretion during two consecutive (2 or 3 hr) periods of incubation. Incubation in 10 or 100 ng/ml TRH for 2 or 3 hr significantly augmented release of TSH bioactivity in a dose-dependent manner. Pituitaries taken from goitrogen (100 micrograms methimazole/day for 10 days)-treated lizards had elevated basal TSH secretion but did not respond to TRH. TRH injection in vivo (5 micrograms/hr for 10 hr) appeared to stimulate acute release of MSH activity as judged by darkening of skin color after each injection, and plasma T4 was significantly elevated at the end of treatment. These results provide additional evidence that the reptilian thyrotrope has functional TRH receptors and the TSH-stimulating activity of the tripeptide along with its effects on other pituitary cells was present at an early stage of reptilian evolution.