The effect of thyroid hormones and thyrotropin releasing hormone (TRH) on prolactin (PRL) secretion has been studied using a primary calf anterior pituitary cell culture system. After mechanical and enzymatic dispersion, cultured pituitary cells were preincubated with T3 or T4 for 48 hr prior to a 24 hr experimental incubation. T3 stimulated the release of PRL into the medium in a dose-related fashion, with an ED50 of 3 nM; at 10 nM T3, a maximal 52 +/- 5% stimulation (p less than 0.001) was observed. T4 at 100 nM stimulated medium PRL 27 +/- 10% (p less than 0.05); the ED50 for T4 was 20 nM. Neither T3 nor T4 affected intracellular PRL content. The stimulation of medium PRL by T3 was observed in medium containing 10% euthyroid as well as 10% charcoal-stripped hypothyroid calf serum. The relative stimulation by TRH of PRL release into the medium was significantly diminished by 10 nM T3 in euthyroid and stripped hypothyroid serum medium, but only as a consequence of the stimulation of basal medium PRL by T3; there was no change in maximal TRH-stimulated PRL release. In medium supplemented with unstripped hypothyroid serum, however, T3 did decrease absolute TRH-stimulated PRL release.