Autoantibodies targeting the proliferating cell nuclear antigen have been considered as a specific biomarker for systemic lupus erythematosus, and were historically identified by indirect immunofluorescence and then confirmed by other more specific immunoassays. Our objective was to investigate the anti-PCNA immune response in various disease conditions. Unselected sera referred to a clinical diagnostic laboratory and other sera from various diseases cohorts and controls were tested for anti-PCNA antibodies by enzyme-linked immunosorbent assay (ELISA), line immunoassay (LIA) and an addressable laser bead assay (ALBIA) using full-length human proliferating cell nuclear antigen. Two out of 2500 sequential, unselected sera (0.07%) referred to a diagnostic laboratory for autoantibody analysis showed a proliferating cell nuclear antigen-like staining pattern. Good agreement was found between ELISA, ALBIA and LIA. At cut-off values resulting in 100% specificity, 52.5% (ELISA), 42.5% (ALBIA) and 35% (LIA) of samples with a proliferating cell nuclear antigen-like indirect immunofluorescence staining pattern were positive. In the indirect immunofluorescence proliferating cell nuclear antigen immunoblot (IB)-positive group, anti-PCNA antibodies were frequently accompanied by anti-Ro52, and in the indirect immunofluorescence PCNA-negative but LIA PCNA-positive group by various other autoantibodies. The prevalence of anti-PCNA antibodies was highest in Sjögren's syndrome (5.0%). In conclusion, the proliferating cell nuclear antigen-like staining pattern was rarely found (0.07%) in sequential, unselected sera. Further, indirect immunofluorescence is not an accurate screening method to identify anti-PCNA antibodies as their presence may be masked by other autoantibodies. The specific association of anti-PCNA antibodies with systemic lupus erythematosus was not confirmed in our study.