We have identified a novel antinuclear antibody (ANA) in the sera of patients with primary biliary cirrhosis (PBC). Sera from 22 of 50 PBC patients stained the nuclei of HEp-2 cells in a pattern of 5-20 discrete dots, on indirect immunofluorescence. Typically, the titers were higher than 1:1,280. This staining pattern was seen in only 4 of 1,240 ANA-positive samples from patients with other connective tissue diseases, and in none of the ANA-positive sera from 49 patients with other liver diseases. Sera which stained the nuclear dots bound a 95-kd polypeptide in immunoblots of HeLa nuclei and in immunoprecipitates of 35S-methionine-labeled HeLa extracts. Affinity-purified antibodies from the 95-kd protein reproduced the nuclear dot immunofluorescence staining pattern, demonstrating that this polypeptide is located in the nuclear dots. ANA of this type, therefore, occur in 44% of patients with PBC, and they appear to be specific for PBC. These antibodies bind a 95-kd protein that is discrete and is scattered throughout the nucleus.