The APB alpha domain in the amyloid beta-protein precursor (APP) promoter contains a nuclear factor binding domain with the core recognition sequence TCAGCT-GAC. Proteins in nuclear extracts from brain and numerous cell lines bind to this domain and it contributes approximately 10-30% to the basal APP promoter activity. Included in this domain is the CANNTG motif, which is recognized by basic helix-loop-helix transcription factors. The same motif is also present in the CDEI element of the yeast centromere and in the adenovirus major late promoter (AdMLP). Here we present evidence based on thermostability, relative binding affinity, eletrophoretic mobility and antibody recognition that the cellular proteins that bind to the APB alpha and CDEI motifs are USF. However, the relative binding affinity for the motifs is different. The affinity of USF for AdMLP is approximately 20-fold higher than for the APB alpha sequence and 5-fold higher than for the CDEI sequence. Mutational analysis suggested that the primary determinant for USF binding affinity resides within the octamer CAGCTGAC, which is composed of the E-box consensus sequence CANNTG followed by the dinucleotide AC. The human homolog of the mouse CDEI binding protein did not bind to either the CDEI sequence or APB alpha.