Antibodies to mammalian cell nucleoli occur spontaneously in certain autoimmune human diseases and have been demonstrated by immunofluorescence. In this study, nucleoli were isolated from fresh calf thymus and antinucleolar antibodies were used as reagents to identify a reactive nucleolar antigen. The antigen was a low molecular weight 4–6S RNA, and the precipitating activity of this antigen was destroyed by treatment with spleen phosphodiesterase. Sera were selected for this study because of predominance of nucleolar staining by immunofluorescence and were found to be from patients with scleroderma, Sjogren's syndrome, and undiagnosed illnesses manifesting Raynaud's phenomenon.