A binding site for the transcription factor Sp1 was identified in the hepatitis B virus (HBV) large surface antigen promoter between nucleotides -49 and -29 by DNase I footprinting analysis with purified recombinant Sp1 protein. Gel retardation analysis using Huh7 nuclear extracts demonstrated that formation of complexes between this sequence element and DNA-binding proteins was specifically inhibited by the HBV major surface antigen and nucleocapsid promoter Sp1 binding sites, as well as by the Sp1 consensus recognition sequence. In addition, gel supershift analysis showed that this sequence element bound factor(s) present in Huh7, HeLa S3, and HepG2.1 cell nuclear extracts which were completely supershifted by the Sp1 antibody and appeared to be the same or similar to the factor(s) which bound the consensus Sp1 recognition sequence. The function of the large surface antigen promoter Sp1 recognition sequence was examined by transient transfection analysis in Drosophila melanogaster Schneider line-2 (SL2) cells. In the context of a minimal promoter element, this Sp1 site was able to mediate transcriptional transactivation by exogenously expressed Sp1. These results suggest that the HBV large surface antigen promoter contains a functional Sp1 binding site which may be involved in the coordinate regulation of HBV transcription by the ubiquitous transcription factor Sp1.