A complex of three proteins (of 80, 70, 58kDa—p80, p70, and p58, respectively) with the ability to bind alphoid DNA (α-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the α-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the α-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to α-satDNA in vitro and that this protein is distinct from the lamins.