Connexin32 (Cx32) encodes the predominant gap junction protein expressed by hepatocytes. We investigated the transcriptional control of Cx32 in expressing and nonexpressing rat liver cell lines and hypothesized that a putative hepatocyte nuclear factor-1 (HNF-1) binding site (centered at mp -187) in the liver-active, P1 promoter is essential for transcription of Cx32. HNF-1alpha was expressed by Cx32-expressing rat liver cell lines and bound the promoter at the -187 site, but was not expressed by non-Cx32-expressing hepatic lines. Stable transfection of non-Cx32-expressing WB-F344 rat liver epithelial cells with HNF-1alpha stimulated a transfected Cx32 promoter element (mp -244 to -33), binding of HNF-1alpha to the -187 site, and expression of endogenous Cx32. Site-directed mutagenesis of this HNF-1 binding site abolished HNF-1alpha binding and proximal promoter activity. Hepatic Cx32 expression was also significantly decreased in HNF-1alpha(-/-) mice. These data indicate that HNF-1alpha is a positive regulator of Cx32 expression in hepatic cells.