Hepatocyte nuclear factor 4α (HNF4α) is essential for the establishment and maintenance of liver-specific gene expression. The HNF4α gene codes for several isoforms whose developmental and physiological relevance has not yet been explored. HNF4α1 and HNF4α7 originate from different promoters, while alternative splicing in 3′ leads to HNF4α2 and HNF4α8, respectively. HNF4α7/α8 were abundantly expressed in embryonic liver and fetal-like hepatoma cells. HNF4α1/α2 transcripts were up-regulated at birth and represented the only isoforms in adult-like hepatoma cells. In line with its expression profile, HNF4α7 activated more avidly than HNF4α1 reporter plasmids for genes that are expressed early. The expression patterns of both isoforms together with the differences observed in their transcriptional activities provide elements accounting for fine-tuning of the activity of HNF4α. The sequential expression of HNF4α7/α8 and HNF4α1/α2 during mouse liver development is the only modification in liver-enriched transcription factors thus far recorded, which parallels the transition from the fetal to the adult hepatic phenotype.