We have examined cytosolic phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) gene transcription and steady-state mRNA expression in chick embryo liver in vivo both constitutively and in response to dexamethasone, cAMP, retinoic acid, and the protein synthesis inhibitors, cycloheximide and pactamycin. We report here that PEPCK mRNA is constitutively expressed between 12 and 16 days of development. PEPCK expression was also transiently but highly inducible on Day 14 by dexamethasone, cAMP, cycloheximide, and pactamycin, but not by retinoic acid. Cycloheximide pretreatment had an additive or synergistic effect on the induction by dexamethasone and/or cAMP. Dexamethasone- and cycloheximide-induced increases in mRNA expression were principally due to increases in the rate of PEPCK gene transcription. Following an initial induction by dexamethasone, PEPCK expression was no longer responsive to a second administration of dexamethasone but was still responsive to cycloheximide and cAMP. PEPCK induction by dexamethasone or cycloheximide progressively diminished between 12 and 15 days of development. By Day 16, PEPCK expression was no longer responsive to dexamethasone, but was still inducible by cAMP and this induction was increased by cycloheximide. These results indicate that PEPCK is transiently inducible by glucocorticoids in chick embryo liver and that there are two developmental switches to the adult phenotype between Days 14 and 16 of development and between Day 16 and 4 days posthatching. Our results also suggest the presence of a developmentally regulated repressor of PEPCK gene expression in chick embryo liver.