We have previously isolated the human cathepsin B promoter and shown that Sp1 and Ets factors are involved in the regulation of cathepsin B expression. Using mutagenesis, transient transfection and electrophoretic mobility shift assays (EMSAs), we further identified regulatory factors that mediate cathepsin B transcription in U87 human glioblastoma cells. An E-box element (CACGTG) adjacent to the transcription initiation site (at nucleotides -7 to -2) was found to be indispensable for cathepsin B promoter activity. Mutation of this E-box element in both pSCB2, a promoter construct with high promoter activity, and pSCB6, a construct with basal promoter activity, led to a 90% decrease in promoter activity in U87 cells. EMSAs demonstrated that upstream stimulatory factor 1 (USF-1) and upstream stimulatory factor 2 (USF-2) bound to the E-box as a heterodimer. Chromatin immunoprecipitation assays revealed that both USF-1 and USF-2 were associated with the cathepsin B promoter. The roles of USF-1 and USF-2 in the regulation of cathepsin B expression were demonstrated by (i) co-transfection experiments showing that USF-1 or USF-2 increased promoter activity by 2.5-fold individually and by 3.4-fold together; (ii) co-transfection of pSCB6 with pUSF-2deltaN (a dominant negative USF-2 expression plasmid) resulting in an 80% decrease in promoter activity; and (iii) mutation of the E-box element (from 5'-CACGTG to 5'-CGCGTT in the pSCB6 basal promoter construct) abolishing transactivation of cathepsin B by USF-1 and USF-2. These results collectively indicate that an E-box at nucleotides -7 to -2 of the cathepsin B promoter is critical to the expression of cathepsin B and that binding of USF-1 and USF-2 to this E-box can regulate cathepsin B promoter activity.