Hepatocyte nuclear factor 4 (HNF4) was first identified as a DNA binding activity in rat liver nuclear extracts. Protein purification had then led to the cDNA cloning of rat HNF4, which was found to be an orphan member of the nuclear receptor superfamily. Binding sites for this factor were identified in many tissue-specifically expressed genes, and the protein was found to be essential for early embryonic development in the mouse. We have now isolated cDNAs encoding the human homolog of the rat and mouse HNF4 splice variant HNF4α2, as well as a previously unknown splice variant of this protein, which we called HNF4α4. More importantly, we also cloned a novel HNF4 subtype (HNF4γ) derived from a different gene and showed that the genes encoding HNF4α and HNF4γ are located on human chromosomes 20 and 8, respectively. Northern (RNA) blot analysis revealed that HNF4γ is expressed in the kidney, pancreas, small intestine, testis, and colon but not in the liver, while HNF4α RNA was found in all of these tissues. By cotransfection experiments in C2 and HeLa cells, we showed that HNF4γ is significantly less active than HNF4α2 and that the novel HNF4α splice variant HNF4α4 has no detectable transactivation potential. Therefore, the differential expression of distinct HNF4 proteins may play a key role in the differential transcriptional regulation of HNF4-dependent genes.