The p53 promoter has been shown to contain a number of potential regulatory motifs. It was previously reported that the upstream stimulating factor (USF) played a central role in regulating the p53 expression. The USF binding site, E-box, is located around 40 bp upstream of the major transcription start site. In this study, it was confirmed that the E-box binds to proteins by DNase I footprinting assay. In the electrophoretic mobility shift assay (EMSA), two retarded bands were detected. One band was abolished by the competition of USF consensus oligonucleotide, but the other band was not. This result indicated that a factor, other than USF, was bound to the E-box. The molecular masses of the binding proteins were determined by a Southwestern-blotting assay. As a result, 46- and 80-kDa proteins were detected. The 46-kDa protein was eliminated by the competition of USF consensus oligonucleotide. Also, the Southwestern-blotting assay with 32P-labeled USF consensus oligonucleotide showed only a 46-kDa protein. Therefore, the 46-kDa protein was USF. These results showed that USF and the 80-kDa protein were bound to the E-box. In addition, it was proved by in vitro transcription assay that this 80-kDa protein had a basal transcriptional activity.