This study investigated the overall clinical impact of anti-alpha-actinin antibodies in patients with pre-selected autoimmune diseases and in a random group of anti-nuclear antibody (ANA)-positive individuals. The relation of anti-alpha-actinin antibodies with lupus nephritis and anti-double-stranded DNA (anti-dsDNA) antibodies represented a particular focus for the study. Using a cross-sectional design, the presence of antibodies to alpha-actinin was studied in selected groups, classified according to the relevant American College of Rheumatology classification criteria for systemic lupus erythematosus (SLE) (n = 99), rheumatoid arthritis (RA) (n = 68), Wegener's granulomatosis (WG) (n = 85), and fibromyalgia (FM) (n = 29), and in a random group of ANA-positive individuals (n = 142). Renal disease was defined as (increased) proteinuria with haematuria or presence of cellular casts. Sera from SLE, RA, and Sjøgren's syndrome (SS) patients had significantly higher levels of anti-alpha-actinin antibodies than the other patient groups. Using the geometric mean (+/- 2 standard deviations) in FM patients as the upper cutoff, 20% of SLE patients, 12% of RA patients, 4% of SS patients, and none of the WG patients were positive for anti-alpha-actinin antibodies. Within the SLE cohort, anti-alpha-actinin antibody levels were higher in patients with renal flares (p = 0.02) and correlated independently with anti-dsDNA antibody levels by enzyme-linked immunosorbent assay (p < 0.007) but not with other disease features. In the random ANA group, 14 individuals had anti-alpha-actinin antibodies. Of these, 36% had SLE, while 64% suffered from other, mostly autoimmune, disorders. Antibodies binding to alpha-actinin were detected in 20% of SLE patients but were not specific for SLE. They correlate with anti-dsDNA antibody levels, implying in vitro cross-reactivity of anti-dsDNA antibodies, which may explain the observed association with renal disease in SLE.