Hepatocyte nuclear factor 1 (HNF-1) is a transcriptional regulatory protein possibly involved in the activation of many liver-specifically expressed genes. HNF-1 mRNA is restricted to a small number of tissues, suggesting that the HNF-1 gene itself is regulated at the transcriptional level. We have isolated and characterized the promoter region of this gene and have determined its transcriptional potential in several cell types by cell-free transcription and transient transfection experiments. In in vitro transcription assays, an HNF-1 promoter is active in nuclear extracts from liver and kidney, two tissues that contain HNF-1, but silent in nuclear extracts from spleen and lung, which are devoid of this transcription factor. Likewise, in transfection experiments, HNF-1 promoter-chloramphenicol acetyltransferase (CAT) fusion genes are expressed in Hep G2 cells, which express HNF-1, but not in mouse L cells or Hela cells, which do not express HNF-1. In both cell-free transcription and transient transfection assays, a relatively short promoter segment located between positions -82 and -40 is necessary and sufficient to direct cell type-specific HNF-1 transcription. This region contains a single site for a DNA-binding protein that has been tentatively identified as hepatocyte nuclear factor 4, a member of the steroid hormone receptor family.