The liver-enriched transcription factor C/EBPα has been implicated in the regulation of numerous liver-specific genes. It was previously reported that mice carrying a homozygous null mutation at the c/ebpα locus died as neonates due to the absence of hepatic glycogen and the resulting hypoglycemia. However, the lethal phenotype precluded further analysis of the role of C/EBPα in hepatic gene regulation in adult mice. To circumvent this problem, we constructed a conditional knockout allele of c/ebpα by using the Cre/loxP recombination system. Homozygous c/ebp-loxP mice, (c/ebpα^fl/fl;fl, flanked by loxP sites) were found to be indistinguishable from their wild-type counterparts. However, when Cre recombinase was delivered to hepatocytes of adult c/ebpα^fl/fl mice by infusion of a recombinant adenovirus carrying the cre gene, more than 80% of the c/ebpα^fl/fl genes were deleted specifically in liver and C/EBPα expression was reduced by 90%. This condition resulted in a reduced level of bilirubin UDP-glucuronosyltransferase expression in the liver. After several days, the knockout mice developed severe jaundice due to an increase in unconjugated serum bilirubin. The expression of genes encoding phosphoenolpyruvate carboxykinase, glycogen synthase, and factor IX was also strongly reduced in adult conditional-knockout animals, while the expression of transferrin, apolipoprotein B, and insulin-like growth factor I genes was not affected. These results establish C/EBPα as an essential transcriptional regulator of genes encoding enzymes involved in bilirubin detoxification and gluconeogenesis in adult mouse liver.