Recently, we have observed that the association of anti-F-actin autoantibodies (Abs) with anti-α-actinin Abs (double reactivity) was correlated with liver disease activity in autoimmune hepatitis type 1 (AIH-1; Guéguen et al., J Clin Immunol 26:495–505, 2006). As anti-α-actinin Abs are also associated with anti-single-stranded (ss) DNA in 16 of 50 (32%) AIH-1 patients, compared with 16 of 401 (4.0%) liver disease control patients, we sought to characterize these Abs in AIH-1. When associated, anti-ssDNA Abs cross-react with anti-α-actinin Abs, but neither with anti-double-stranded DNA Abs nor with anti-F-actin Abs. Furthermore, anti-α-actinin Abs are associated with high-avidity anti-ssDNA Abs in AIH-1. In addition, double reactivity against α-actinin and ssDNA was correlated with clinical and histological activity of the disease and untreated AIH-1 patients.