The gene for phosphoenolpyruvate carboxykinase (PEPCK), a target of CCAAT/enhancer-binding protein-α (C/EBPα) and -β (C/EBPβ), begins to be expressed in the liver at birth. Mice homozygous for a deletion in the gene for CEBPα (C/EBPα−/−mice) die shortly after birth of hypoglycemia, with no detectable hepatic PEPCK mRNA and negligible hepatic glycogen stores. Half of the mice homozygous for a deletion in the gene for CEBPβ (C/EBPβ−/−mice) have normal glucose homeostasis (phenotype A), and the other half die at birth of hypoglycemia due to a failure to express the gene for PEPCK and to mobilize hepatic glycogen (phenotype B). Insulin deficiency induces C/EBPα and PEPCK gene transcription in the livers of 19-day fetal rats, whereas dibutyryl cyclic AMP (Bt2cAMP) increases the expression of the gene for C/EBPβ and causes a transient burst of PEPCK mRNA. Bt2cAMP induces PEPCK mRNA in the livers of fetal C/EBPα−/−mice, but at only 20% of the level of control animals; however, there is no induction of PEPCK mRNA if the cyclic nucleotide is injected into C/EBPα−/−mice immediately after delivery. The expression of the gene for C/EBPβ is markedly induced in the livers of C/EBPα−/−mice within 2 h after the administration of Bt2cAMP. C/EBPβ−/−mice injected at 20 days of fetal life with Bt2cAMP have a normal pattern of induction of hepatic PEPCK mRNA. In C/EBPβ−/−mice with phenotype B, the administration of Bt2cAMP immediately after delivery induces PEPCK mRNA, causes the mobilization of hepatic glycogen, and maintains normal glucose homeostasis for up to 4 h (duration of the experiment). We conclude that C/EBPα is required for the cAMP induction of PEPCK gene expression in the liver and that C/EBPβ can compensate for the loss of C/EBPα if its concentration is induced to appropriate levels.