The homeodomain-containing transcription factor hepatocyte nuclear factor 1 (HNF-1) most likely plays an essential role during liver organogenesis by transactivating a family of greater than 15 predominantly hepatic genes. We have isolated cDNA clones encoding mouse HNF-1 and expressed them in monkey COS cells and in the human T-cell line Jurkat, producing HNF-1 DNA-binding activity as well as transactivation of reporter constructs containing multimerized HNF-1 binding sites. In addition, the HNF-1 gene was assigned by somatic cell hybrids and recombinant inbred strain mapping to mouse chromosome 5 near Bcd-1 and to human chromosome 12 region q22-qter, revealing a homologous chromosome region in these two species. The presence of HNF-1 mRNA in multiple endodermal tissues (liver, stomach, intestine) suggests that HNF-1 may constitute an early marker for endodermal, rather than hepatocyte, differentiation. Further, that HNF-1 DNA-binding and transcriptional activity can be conferred by transfecting the HNF-1 cDNA into several cell lines indicates that it is sufficient to activate transcription in the context of ubiquitously expressed factors.