The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T 3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization,(ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T 3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH 3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T 3 stimulation of rGH promoter activity is dependent on the addition of T 3 to the GH 3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T 3, but not T 4 or reverse T 3, specific. T 3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to-160) but not with Pit-1/GHF-1 (-137 to-65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T 3 effect. These results provide evidence that T 3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T 3 might involve direct interaction of TRs with the general transcriptional apparatus.