Objective. Although anti-double-stranded DNA (anti-dsBNA) antibodies are important in lupus nephritis, the question regarding which glomerular structures (α-actinin, nucleosomes, or others) are recognized by nephritogenic anti-dsDNA antibodies is still controversial. In this study, we determined which glomerular structures are recognized by monoclonal and in vivo-bound nephritogenic antibodies. Methods. Western blotting was used to analyze the ability of nephritogenic anti-dsDNA antibodies to recognize glomerular and nucleosomal structures. Sera from patients with lupus nephritis, sera from random antinudear antibody-positive patients, and paired antibodies from sera and kidney eluates from nephritic (NZB × NZW)F1 mice were analyzed for activity against proteins identified by monoclonal nephritogenic antibodies, and against α-actinin, dsDNA, nucleosomes, histone H1, heparan sulfate, DNase I, and type IV collagen. Immunoelectron microscopy was used to determine the glomerular localization of α-actinin and in vivo-bound autoantibodies in nephritic (NZB × NZW)F1 mouse kidneys. Results. Anti-α-actinin antibodies were observed in human and murine lupus nephritis sera and in sera from patients without systemic lupus erythematosus and were not detected in kidney eluates from nephritic mice. Antibodies to dsDNA and histone H1 were detected in all eluates. Western blot analyses revealed that nephritogenic anti-dsBNA antibodies recognized a 32-kd band, identified as histone H1. Competitive enzyme-linked immunosorbent assay demonstrated that nephritogenic monoclonal antibodies, and dominant antibodies eluted from nephritic kidneys, cross-reacted with dsDNA and H1. This cross-reactive anti-H1 specificity was largely absent in sera from those mice. Immunoelectron microscopic analysis of nephritic (NZB × NZW)F1 mouse kidneys revealed that antibodies eluted from kidneys, but not anti-α-actinin antibodies, bound to distinct nephritis-associated electrondense structures linked to glomerular basement membranes. Conclusion. Cross-reactive anti-dsDNA/antihistone III antibodies, but not anti-α-actinin antibodies, are central among those deposited in nephritic glomeruli.