TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.