For the identification of SS-B autoantibodies in the sera of patients with rheumatic diseases (n = 319) a sensitive and specific enzyme-linked immunosorbent assay was developed using highly purified human SS-B antigen as a reference antigen. In 30/319 patients' sera SS-B autoantibodies were detected. In this group, the ratio female : male patients was outstandingly high (29:1), the serum of the single male patient exhibiting the lowest titer of SS-B autoantibodies. The highest portion of anti-SS-B positive sera was observed in the group of patients with systemic Lupus erythematosus (48%) followed by the groups with undifferentiated connective tissue disease (17%), Sjögren-syndrome (13%) and rheumatoid arthritis (6%). The protein components reacting with human SS-B autoantibodies were characterized in permanent cell lines of various species. In each of 9 human cell lines only one component with a molecular weight of 49 kD reacted; in cell lines of other species analogously, only one component reacted, showing slightly different molecular weights depending on the species. The SS-B antigen isolated from human cells contained at least 7 isoelectric variants, all reacting with human SS-B autoantibodies.