An acute incubation procedure, using explanted normal rat hemipituitaries pretreated with fresh plasma obtained from pituitary donor animals, was employed to further investigate the in vitro stimulation of prolactin (PRL) release by thyrotropin-releasing hormone (TRH). Pretreatment with dopamine (0.1 µg/ml) caused a 30-50% decrease in the amount of PRL released into incubation media; the inhibitory effect of dopamine was not reversed by treatment with 0.5-6.0 ng. TRH, although these TRH concentrations consistently stimulated PRL release from pituitaries not exposed to dopamine. Treatment with thyroxine (10<sup>-6</sup> to 10<sup>-5</sup> M) showed a competitive inhibition of thyrotropin release by TRH (0.5 ng), but was without effect on TRH-stimulated PRL release. Cycloheximide (100 µg/ml) blocked a net increase in PRL levels. TRH, nevertheless, significantly increased PRL release in the presence of cycloheximide. The results indicate that neither dopamine nor thyroxine compete with TRH in causing PRL release, and that the TRH stimulation of PRL release is unrelated to ongoing levels of hormone synthesis.