Immunoaffinity-purified Ku protein was used to screen sera from patients with systemic lupus erythematosus (SLE), scleroderma, myositis and Sjögren's syndrome for anti-Ku antibodies in a quantitative immunoblot assay. Sixteen percent of the 159 studied sera were reactive with the Ku protein; significantly increased frequencies of anti-Ku antibodies were found in SLE (19%) and scleroderma (14%) sera. Patients with myositis and Sjögren's syndrome showed similar frequencies. All positive sera had antibodies to the 86 kD subunit of Ku protein; only one serum did not react with 70 kD subunit. Frequencies of other autoantibodies were compared in anti-Ku positive and negative patients. Only anti-Sm antibodies, especially in the absence of anti-nRNP, appear to be associated with the presence of anti-Ku antibodies. A strong correlation between anti-Ku antibodies and the class II HLA antigen DQw1 (89% of the positive sera) was observed, suggesting participation of MHC genes in the mounting of the anti-Ku immune response.