The middle and small surface proteins of hepatitis B virus are translated from 5'-heterogeneous transcripts specified by the S promoter. We have generated a series of linker-substitution mutants that encompass the 130 base pairs comprising this promoter and measured the amount of transcripts and protein products synthesized from each mutant. The results confirm our previous finding that a CCAAT element is an important up-stream activating element for this promoter, as mutation of this element leads to a >20-fold decrease in promoter activity. In vitro binding assays showed that the cellular transcription factor NF-Y (CCAAT-binding factor) binds to this element, and expression of a dominant-negative NF-Y subunit in transfected cells specifically reduced surface protein expression from the S promoter via the CCAAT element. In addition, two Sp1 sites also contribute to S promoter activity by a total of approximately 6-fold. Therefore, the S promoter is activated by both NF-Y and Sp1, but more strongly by the former factor.