Satellite DNA (satDNA) is the main component of residual DNA in nuclear matrix (NM) preparations. Gel mobility shift assay (GMSA) revealed specific human satellite 3 (HS3) binding activity in NM extracts. An HS3 binding protein was purified using diethylaminoethyl (DEAE)-cellulose and preparative GMSA. The binding was specific, although other satDNA fragments compete to some extent for the binding. DNase I footprinting and methylation interference revealed multiple points of protection distributed throughout the HS3 fragment with periodicity of about 10 bp, mostly inside an AT island. Polyclonal antibodies (AB) were raised against HS3-protein complexes cut from the preparative GMSA gel. On immunoblots, AB recognise a protein, which is not lamin, with apparent molecular mass 70 kDa, the same as revealed by purification (p70). In in situ nuclear matrix preparations combined immunofluorescence (AB) and fluorescent in situ hybridisation (HS3) shows that HS3 and p70 areas correspond to each other. The localisation of this protein detected with AB in interphase nuclei coincides with the heterochromatic regions which surround nucleoli in correspondence with the known HS3 position in the nuclei.