Following recent reports that pathogenic murine anti-DNA antibodies bind to alpha-actinin, it was obviously of interest to assess the ability of human pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies to bind this antigen. Both human monoclonal anti-DNA antibodies and antibodies affinity purified from the sera of patients with systemic lupus erythematosus (SLE) were investigated. An enzyme-linked immunosorbent assay was established to measure immunoglobulin binding to alpha-actinin. Antibodies binding dsDNA were purified from the sera of SLE patients who either had active renal disease or had never had renal disease. Serum samples were selected at times when the patients' sera exhibited high IgG binding to dsDNA. The binding of supernatants from 3 high-affinity human anti-dsDNA IgG hybridomas (RH14, B3, and DIL-6) and 7 human IgM anti-DNA hybridomas was also investigated. A greater proportion of anti-dsDNA IgG-binding antibodies purified from patients with renal disease bound to alpha-actinin than did those purified from the sera of patients without renal disease. The specificity of binding to the 100-kd alpha-actinin molecule was confirmed by Western blotting. The pathogenic human antibodies RH14 and B3 bound strongly to alpha-actinin, while nonpathogenic DIL-6 bound very weakly. RT84, the IgM antibody that binds dsDNA with the highest affinity, exhibited the greatest binding to alpha-actinin. The results of our study support the findings of previous studies using murine anti-DNA monoclonal antibodies, which suggest that pathogenic anti-dsDNA antibodies cross-react with alpha-actinin.