The precursor peptide of TRH (prepro-TRH) contains five copes of pro-TRH linked by other peptide sequences. These peptides are coprocessed with TRH in the median eminence of the hypothalamus and released into the portal circulation, rendering this family of peptides available to act at the level of the anterior pituitary. Therefore, we tested the potential bioactivity of one cryptic peptide, prepro-TRH amino acids 160-169 [prepro-TRH-(160-169)], in a TRH-responsive pituitary cell line (GH3). In a heterologous TSH expression assay, we found that prepro-TRH-(160-169) stimulated TSHβ gene promoter activity in a time- and dose-dependent manner; moreover, the effect of prepro-TRH-(160-169) was more rapid and of greater magnitude than that of TRH on TSHβ-directed chloramphenicol acetyltransferase synthesis. In the same cells, we found that prepro-TRH-(160-169) stimulated PRL synthesis and secretion, but the effect was similar in magnitude and duration to that of TRH. The effect of prepro-TRH-(160-169) appears to be additive to that of TRH, suggesting that prepro-TRH-(160-169) may act through a mechanism separate from thatofTRH. Thus, prepro-TRH-(160-169) has potent endocrinological effects at the level of the genome.