B-cell-specific activator protein (BSAP) encoded by the Pax5 gene plays a critical role during B-cell development. We have analyzed the 5'-flanking region plus the 5'-untranslated region (5'-UTR) of human Pax5 exon1A to clarify its regulatory mechanisms. Functional dissection of these regions by luciferase reporter assays indicated that a cluster of regulatory elements acts as a strong repressor between +320 and +453. Insertion of this segment between the heterologous simian virus 40 (SV40) promoter and the luciferase gene in both the sense and reverse orientation sharply reduced the luciferase activity, but insertion into the upstream of the SV40 promoter did not. This suggests that this segment must be located in the 5'-UTR to function effectively. A search through databases with the sequence of this segment did not reveal any known DNA binding factor site. Electrophoretic gel mobility shift assay (EMSA) experiments demonstrated that unknown factors bound to the fragment +408 to +429. Insertion of this fragment between the SV40 promoter and the reporter gene strongly suppressed the luciferase activity. Competitive EMSA indicated that the region between nucleotides +413 and +427 encompassed the binding site of the unknown factors and was hence regarded as a repressor element. Mutagenesis in this element significantly recovered reporter gene activity. These results suggest that the segment +320 to +453, especially the repressor element +413 to +427, in the 5'-UTR is involved in the regulation of Pax5 gene expression.