We evaluated the extent to which anti-proteasome activator (PA) 28alpha antibodies act as anti-cytoplasmic antibodies in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). Sera from 46 SLE patients without SS, 11 SLE patients with SS, and 45 primary SS patients were tested. Using anti-PA28alpha and anti-PA28gamma (Ki) antibodies purified from nitrocellulose membranes onto which recombinant PA28alpha and Ki had been transferred, the cellular distributions of the targeted antigens were analyzed immunohistochemically. In addition, the incidence of anti-PA28alpha antibodies was compared with those of other anti-cytoplasmic antibodies. Immunofluorescent staining showed that purified anti-PA28alpha antibodies reacted with the cytoplasm of HEp-2 cells, whereas purified anti-Ki antibodies reacted with nucleoplasm. Among the 15 SLE patients without SS, the six SLE patients with SS, and the 30 primary SS patients who were anti-cytoplasmic-antibody positive, anti-SS-A/Ro antibodies were the most frequently detected (53, 67, and 70%, respectively); anti-PA28alpha antibodies were, respectively, detected in 33, 50, and 40% of those patient groups, incidences that were higher than those of anti-ribosomal P, anti-smooth muscle and anti-mitochondrial M2 antibodies. These results show that anti-PA28alpha antibodies are major anti-cytoplasmic antibodies in patients with SLE and SS, and the distinct cellular distributions of PA28alpha and Ki suggest these proteins are associated with different cellular functions.