SS-B/La, an ubiquitous nuclear protein of 46-48 kD, is a target antigen of autoantibodies in SLE and Sjogren's syndrome and is involved in the maturation of RNA polymerase III transcripts such as 5S RNA and tRNAs. We have previously shown (14, 15) that SS-B consists of two protease-resistant domains of 23 and 28 kD, with the latter containing the RNA binding site. The epitopes of SS-B/La reactive with human autoantibodies are conserved among several mammalian species examined. BALB/c mice immunized with affinity-purified calf thymus SS-B produce IgG anti-SS-B/La antibodies, which reacted with bovine, human, and rabbit SS-B but not with mouse SS-B/La. The spleen of a mouse with the highest antibody titer was selected for fusion with P3 myeloma. Five IgG1k mAbs (A1-5) were selected by ELISA and immunoblotting. All except A3 reacted with the 28-kD domain. A1, A2, and A3 were capable of immuno-precipitating the 48-kD SS-B protein and its associated RNAs. A1, A2, and A3 also gave fine nuclear speckled staining on human, monkey, bovine, and rabbit cells that was similar in appearance to that with human autoantibodies, but in contrast to staining with human autoantibodies, they did not stain cells from rat, mouse, or rat kangaroo. It appears that human autoantibodies target highly conserved epitopes that can be distinguished from epitopes recognized by immunization-induced murine mAbs. Taken together with other data, it appears that human autoantibodies may be recognizing epitopes that are active or catalytic sites of molecules subserving important cellular functions.