Ovarian follicles isolated from rainbow trout during early exogenous vitellogenesis were used to study the in vitro effects of thyroid hormones on salmon gonadotropin (GtH)-induced estradiol-17β (E 2) secretion. Triiodothyronine (T 3) alone did not alter E 2 secretion but T 3 in the presence of GtH (0.5 μg/ml) modified E 2 secretion according to a biphasic dose-response curve. Maximum E 2 secretion occurred at 1.9 × 10 −8 M T 3; a concentration of 3.0 × 10 −7 M was inhibitory. T 3 was more potent in stimulating GtH-induced E 2 secretion than thyroxine. The stimulatory and inhibitory effects of T 3 were consistent over a range of GtH concentrations (0.1–1.0 μg/ml). Cycloheximide (0.1–10 μ M) decreased E 2 secretion by GtH-treated follicles in a dose-dependent manner, but failed to overcome all the stimulatory effects of T 3. Time course studies with follicles incubated with GtH, GtH + T 3, GtH + cycloheximide, orGtH + T 3 + cycloheximide indicated that T 3 stimulation of GtH-induced E 2 secretion occurs within 6 hr. It is concluded that thyroid hormones amplify the effects of GtH on E 2 secretion by isolated ovarian follicles; at least a part of this effect does not require de novo protein synthesis.