Objectives: To determine the prevalence and diagnostic utility of monospecific anti-Ro52 (defined as an immune response against Ro-52 antigen in the absence of reactivity to Ro-60 antigen) reactivity in selected autoimmune diseases. Study design: Stored diagnostic non-consecutive serum samples obtained from patients with systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS), systemic sclerosis, idiopathic inflammatory myopathies (IIM), rheumatoid arthritis, primary biliary cirrhosis and mixed essential cryoglobulinaemia were analysed by line immunoassay to detect the presence of anti-Ro52 and other autoantibodies. Results: Monospecific anti-Ro52 reactivity was found in 51 (12.7%) of the 402 samples tested. Anti-Ro52 was the most common serological marker in patients with IIM (35/147, 23.8%) and co-occurred with anti-Jo1 (10/18, 55.6%; p=0.02). The prevalence of anti-Ro52 reactivity was significantly more than anti-Ro60 reactivity in patients with IIM, systemic sclerosis, primary biliary cirrhosis, mixed essential cryoglobulinemia and pSS. The mean signal intensity of anti-Ro52 reactivity was significantly higher in pSS than SLE and associated with rheumatoid factor positivity. The mean signal intensity of anti-Ro52 correlated with anti-Ro60 and anti-La in pSS and SLE. Conclusions: Monospecific anti-Ro52 reactivity is not disease specific but may be of importance in patients with IIM. Furthermore, as anti-Ro52 reactivity is more prevalent than anti-Ro60 reactivity in certain autoimmune conditions, specific testing for their distinction in clinical practice is recommended.