Hepatic expression of the gene for phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) (EC 4.1.1.32) in birds occurs prior to birth and decreases to negligible levels before hatching, whereas in mammals the gene for PEPCK-C in the liver is expressed at birth and is active throughout the life of the animal. The administration of cyclic AMP to adult chickens results in the induction of transcription of the gene for PEPCK-C and the transient accumulation of PEPCK-C mRNA in the liver. DNase I footprint analysis of 330 bp of the avian PEPCK-C promoter immediately 5′ of the start-site of transcription indicated the presence of several protein binding domains, purified CAAT/enhancer binding protein α, cAMP regulatory element binding protein and nuclear factor-1 bound to these regions of the promoter. Sequences corresponding to an hepatic nuclear factor-1 binding domain and to the insulin response sequence, previously identified in the rat PEPCK-C promoter, were also found in the chicken PEPCK-C promoter. Co-transfection of an expression vector for CAAT/enhancer binding protein α or CAAT/enhancer binding protein β markedly stimulated transcription from both the chicken and rat PEPCK-C promoters in human hepatoma cells. Sequences involved in the regulation of gene transcription by cyclic AMP and insulin were found to reside between −210 and +1 of the avian PEPCK-C promoter. In general, transcription from the avian promoter was more sensitive to inhibition by insulin than was noted for the rat PEPCK-C promoter, which may explain in part the lack of expression of the gene for PEPCK-C in the livers of adult birds.