The plasma transport protein for glucocorticoids, corticosteroid-binding globulin (CBG), is produced by hepatocytes, and expression of the CBG gene (Cbg) in the liver is controlled by a variety of hormones, environmental stimuli, and developmental cues. The rat Cbg proximal promoter, including 145 base pairs (bp) from the transcription start site, contains two cis-regulatory elements (designated P1 and P2), and is as transcriptionally active as a much more extended region (approximately 1.2 kbp) of the promoter. We have now characterized the rat liver nuclear proteins that interact with P1 and P2. Several proteins interacted specifically with P1 during an electrophoretic mobility shift assay (EMSA), and based on ultraviolet (UV) cross-linking and Southwestern blot analyses, as well as an antibody-supershifting EMSA, one of these has been identified as hepatic nuclear factor (HNF)1 beta. The major band shift formed with P2 in an EMSA appears to comprise a protein that migrates as a doublet of 58 and 62 KDa on sodium dodecylsulfate-polyacrylamide gel ectrophoresis (SDS-PAGE) after UV cross-linking with an oligonucleotide containing P2, as well as during Southwestern blot analyses. Mutations of the CCAAT sequence within P2 also prevent binding to this protein, the physicochemical properties of which resemble the CCAAT-binding protein CP2. Functional analyses of this region of the rat Cbg proximal promoter fused to a luciferase reporter gene demonstrated that mutations within P1, which prevent its interaction with NHF1, do not influence adversely its transcriptional activity. Thus, although members of the HNF1 family of nuclear proteins play an essential role in the transcriptional activation of several other related genes (e.g., thyroxin-binding globulin and alphal-antitrypsin) in hepatocytes, HNF1 beta does not appear to be required for the basal activity of the rat Cbg promoter. In addition, deletion of P2 from the proximal promoter abolishes transcriptional activity and the CCAAT-binding protein that interacts with P2 probably represents an important determinant of Cbg expression under different physiological conditions.