The DNA-binding proteins which recognize the regulatory sequence elements of the hepatitis B virus (HBV) major surface antigen promoter were examined by gel retardation analysis, using nuclear extracts from the human hepatoma cell line Huh7. Using this assay, we identified four regions (B, D, E, and F) of the promoter that interact with the same or similar transcription factor(s). In addition, the recognition sequence for the Sp1 transcription factor bound the same or similar transcription factor(s) present in Huh7 cell nuclear extracts, and this binding was inhibited by the four major surface antigen promoter elements, B, D, E, and F. Purified Sp1 transcription factor was shown to bind to three (B, D, and F) of the major surface antigen promoter regulatory sequence elements by DNase I footprinting. Using transient transfection assays with Drosophila Schneider line 2 cells, we found that transcription from the major surface antigen promoter was transactivated by exogenously expressed Sp1, whereas transcription from the other three HBV promoters was not. Deletion analysis of the major surface antigen promoter demonstrated that the promoter region between -35 and +157 was sufficient to confer Sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor. The function of the B, D, E, and F promoter elements was further examined by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. This finding demonstrates that the HBV major surface antigen promoter contains four functional Sp1 binding sites which probably contribute to the level of expression from this promoter during viral infection.