Counterimmunoelectrophoresis (CIE), RNA precipitation, ELISA and immunoblotting against cytoplasmic HeLa cell extract (IB-HeLa) and erythrocyte extract (IB-RBC) were applied to detect anti-Ro(SSA) antibodies in 93 sera selected from patients with various autoimmune diseases [47 were anti-Ro(SSA) positive by CIE]. The RNA precipitation assay, which demonstrated the highest sensitivity was selected as the reference method. CIE was found to be reliable with a specificity of 100% and a sensitivity of 89%. ELISA showed a comparable specificity (95%) but somewhat lower sensitivity (72%). Antibodies to 52 or 60 kDa Ro(SSA) proteins by IB-HeLa demonstrated a high specificity (95 and 97% respectively) but a low overall sensitivity (36 and 17% respectively). Anti-Ro(SSA) antibodies to 52, 54 and 60 kDa erythrocyte proteins by IB-RBC, had a variable overall specificity (95, 97 and 57%) and sensitivity (51, 13 and 34%). The anti-52 kDa antibodies detected by IB-HeLa correlated to those found by IB-RBC (P < 0.001) and occurred predominantly in primary Sjögren's syndrome (P < 0.001, sensitivity: 71 and 77%) as well as in sera with anti-Ro(SSA) and anti-La(SSB) antibodies (P < 0.001). These findings confirm that RNA precipitation assay has the highest sensitivity and specificity for anti-Ro(SSA) antibody detection. However, until a more sensitive ELISA is available, CIE because of its reliability appears to be the method of choice. Finally IB-RBC was found to be more sensitive than IB-HeLa for the detection of anti-Ro52 kDa antibodies.