CCAAT/enhancer binding protein (C/EBP) family members are known to transactivate the gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) in hepatocytes via promoter proximal C/EBP response elements. PEPCK is also expressed in adipocytes; however, fibroblasts that are homozygous null for C/EBP beta cannot express PEPCK when induced to differentiate into adipocytes (Tanaka et al, EMBO J. 16, 7432-7443, 1997). This along with our previous observation that an upstream adipocyte-specific enhancer contains multiple putative C/EBP binding elements suggested the possibility that C/EBP beta transactivates the PEPCK gene in adipocytes via distal elements. We report here that C/EBP beta transactivates a PEPCK-luciferase chimera in transient transfection assays. C/EBP beta acted independently of peroxisome proliferator-activated receptor gamma (PPAR gamma) which is required for function of the enhancer. C/EBP beta in nuclear extracts and recombinant C/EBP beta bound three of the putative C/EBP-binding elements within the enhancer. C/EBP beta binding to these three elements was strongly cooperative. However, mutation of all three elements did not affect reporter transactivation by C/EBP beta suggesting that additional elements participate in PEPCK regulation or that the effects of C/EBP beta are indirect.