Transcription of the gene for phosphoenolpyruvate carboxy-kinase (PEPCK) is stimulated by thyroid hormone (T3), glucagon (via cyclic AMP) and glucocorticoids. A region of the PEPCK promoter between -332 and -308 mediates the induction of transcription by T3. To characterize this region further, mutations were introduced into this region of the PEPCK promoter and the modified promoters ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. Using these PEPCK-CAT vectors in transient transfections in HepG2 cells, it was found that T3 stimulates PEPCK transcription through two direct repeats of the AGGTCA motif located between nucleotides -330 and -319 [PEPCK-thyroid-hormone-responsive element (TRE)]. The beta form of the T3 receptor (TR beta) bound PEPCK-TRE as a homodimer but bound far more efficiently as a heterodimeric complex with the retinoid X receptor (RXR). An additional region called P3(I) (-250 to -234) is required for T3 responsiveness and binds members of the CCAAT-enhancer-binding protein (C/EBP) family. P3(I) contains an AGGTCA-like motif that can bind the TR beta-RXR heterodimer. Mutagenesis of this motif abolished TR beta-RXR binding without reducing T3 induction. Mutation of the C/EBP-binding site or insertion of a cyclic AMP-responsive-binding-protein site at P3(I) eliminated the T3 response. Our results indicate that T3 stimulation of PEPCK transcription is mediated by TR beta bound to PEPCK-TRE and requires C/EBP to be bound at the P3(I) site.