A full-length cDNA encoding human 60-kDa Ro/SS-A protein was transfected into and overexpressed in the human cell line HEp-2, with the aim of developing an improved reagent for indirect immunofluorescence (IF) detection of anti-Ro autoantibodies. Stable transfectants were analyzed by IF using a panel of 20 precipitin-positive anti-Ro human sera. Transfectants showed bright finely speckled nuclear and nucleolar staining. No surface membrane expression was detected despite marked overexpression of 60-kDa Ro. All human anti-Ro sera reacted with the transfectants with titers ranging from 1:320 to 1:40,960. The same sera tested on untransfected cells showed titers from negative (3 sera) to 1:160. These transfectants dramatically increase the sensitivity of IF testing (mean increase in titer of 41-fold) and allow detection of specific anti-Ro antibodies in samples negative or equivocal on untransfected cells. The staining patterns of antisera of other important specificities remained unaltered. In a study of sera from 22 patients with systemic lupus erythematosus, anti-60-kDa Ro autoantibodies were detected in all sera when tested on 60-kDa Ro-transfected HEp-2 cells; however, in 12 of 22 sera autoantibodies were undetectable by recombinant 60-kDa Ro ELISA. The 60-kDa Ro transfectants are a simple and sensitive method for detection of anti-Ro antibodies in patients with systemic rheumatic diseases.