We have used the gel retardation assay to investigate the binding of nuclear proteins to the duplicated CACCC boxes in the β-globin gene promoter region. The effect of β-thalassemia mutations affecting both of these consensus sequences (the -88 C→T and -101 C→T mutations) were studied by using appropriate mutant oligonucleotides. Upon incubation with nuclear proteins from human erythroleukemia cells, the synthetic oligonucleotides containing single and/or duplicated CACCC box(es) generated five retarded bands. Bands B1 and B2 appeared to be highly specific complexes as determined by competition experiments whereas bands B3, B4 and B5 were nonspecific. The binding of the specific trans-acting factors was stable and could be competed out only by relatively high concentrations of competitor DNA. The proximal CACCC box (as in the -88N probe) appeared to be essential in the binding of nuclear proteins and a mutation at nt -88 (C→T) abolished binding completely. In contrast, the distal CACCC box showed no binding activity either as normal (-101N) or as mutant (-101M). A full competition with the Sp1 oligonucleotide, even at low concentrations, suggested a high affinity binding to the Sp1 consensus sequence. The close similarities in the binding patterns of the labelled -88N, -88N/-101N and that of the labelled Sp1 probe add further credence to the possibility that bands B1 and B2 may be due to the Spl protein, a ubiquitously expressed trans-activator.