The hepatitis B virus nucleocapsid minimal promoter contains sequence elements which are similar to the Sp1 transcription factor binding site consensus sequence. The interaction of these regulatory elements with Sp1 was examined by DNase I footprinting with purified Sp1 protein and DNase I footprinting and gel retardation analysis with nuclear extracts from human cell lines and was examined functionally with transient transfection assays in human hepatoma and Drosophila melanogaster Schneider line-2 cells. DNase I footprinting identified two regions of the nucleocapsid promoter, representing three recognition elements, that bound purified Sp1. Gel retardation analysis with Huh7 nuclear extracts demonstrated that each of the three recognition elements bound the same or similar transcription factor(s) as that recognized by the Sp1 consensus sequence recognition element. The function of the nucleocapsid promoter elements was examined by transient transfection assays in D. melanogaster Schneider line-2 cells 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. In addition, the second Sp1 site was shown to be an essential element of the nucleocapsid promoter in human hepatoma cells. This demonstrates that the hepatitis B virus nucleocapsid promoter contains three functional Sp1 binding sites which may contribute to the level of transcription from this promoter during viral infection.