Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene is repressed during fetal liver development and activated at birth. It has been shown that the PEPCK gene is a retinoid-responsive gene, but whether it is regulated by vitamin A in the fetus has not been established. In this study, we found that PEPCK mRNA can be detected in the murine fetal liver as early as gestational d 17. In addition, expression and cAMP induction of the PEPCK gene during late gestation and at birth require vitamin A sufficiency in the fetus and neonate. The PEPCK promoter contains several regulatory elements that bind a diverse array of transcription factors and nuclear coregulators, although it is largely unknown which of these factors are expressed early in liver development. Expression of some of these nuclear factors in livers of fetal mice was investigated by immunohistochemistry (IHC). Fetuses were from dams that were fed from the beginning of gestation diets that were adequate or devoid of vitamin A. Hepatocyte nuclear factor 4alpha (HNF4alpha) was expressed at the earliest stage of liver development on d 11, whereas retinoid X receptor alpha (RXRalpha) and nuclear coactivator CREB-binding protein (CBP) were expressed from d 16 onward. Although expressions of RXRalpha and CBP in livers of vitamin A-sufficient and vitamin A-depleted fetal mice did not differ, the level of HNF4alpha was consistently lower in the latter. Our findings strongly suggest that vitamin A is required during liver development for staged expression of the PEPCK gene and that HNF4alpha may be involved in mediating vitamin A regulation of the PEPCK gene at these critical periods.