To study in more detail the cross-reactive binding of anti-DNA antibodies to heparan sulfate (HS) and heparan sulfate proteoglycan (HSPG) purified from glomerular basement membranes (GBM), the binding pattern of 31 murine IgG anti-DNA MoAbs, derived from MRL/lpr, NZB/W and graftversus-host diseased mice, was analysed. In ELISA we found binding of 10 anti-DNA MoAbs to HS. Seven of the 10 anti-HS positive clones bound to HSPG but not to the HSPG core protein in ELISA and/or on Western blots. However, DNase-I treatment partly reduced this binding, whereas after purification of MoAb by protein-A sepharose chromatography under dissociative conditions, all clones completely lost their binding capacity to HS and HSPG. Culturing of hybridoma cells in the presence of ³H-thymidine revealed DNA bound to the MoAb. Although the binding to HS and HSPG could be reconstituted by the addition of the protein-A column effluent, this was not possible by the addition of DNA alone. Therefore, we performed immunoprecipitation of the effluent with purified MoAb and subsequent SDS-PAGE which showed that the complex also contained histones. However, histones alone were also not able to reconstitute the binding to HS and HSPG. It is concluded that binding of anti-DNA MoAb to HS and GBM-HSPG is mediated via bound complexes containing both DNA and histones. A comparable reaction with polyclonal anti-DNA Ab might play a role in the pathogenesis of SLE nephritis, since histones have a very high affinity for HS, the major glycosaminoglycan of the GBM.