Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by thyroid hormone (T3) and cAMP. Two DNA elements in the PEPCK promoter are required for T3 responsiveness including a thyroid hormone response element and a binding site called P3(I) for the CCAAT enhancer-binding protein (C/EBP). Both the alpha and beta isoforms of C/EBP are highly expressed in the liver. C/EBPalpha contributes to the liver-specific expression and cAMP responsiveness of the PEPCK gene. In this study, we examined the ability of C/EBPbeta when bound to the P3(I) site to regulate PEPCK gene expression. We report that C/EBPbeta can stimulate basal expression and participate in the induction of PEPCK gene transcription by T3 and cAMP. The cAMP-responsive element-binding protein and AP1 proteins that contribute to the induction by cAMP are not involved in the stimulation by T3. A small region of the transactivation domain of C/EBPbeta is sufficient for the stimulation of basal expression and cAMP responsiveness. Our results suggest that C/EBPalpha and C/EBPbeta are functionally interchangeable when bound to the P3(I) site of the PEPCK promoter.