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The detection of anti-cyclic citrullinated peptide (anti-CCP) IgG antibodies in blood is mainly used for the diagnosis of rheumatoid arthritis. Falsely elevated anti-CCP IgG antibodies due to anti-streptavidin IgG antibodies were suspected in our laboratory.In this study, we evaluated, in a standardized approach, the prevalence of anti-streptavidin IgG antibodies in a primary care setting and the effect of anti-streptavidin IgG antibodies on anti-CCP IgG assays from three different important commercial manufacturers (Abbott, Roche Diagnostics, Thermo Fisher Scientific). Three different populations were consecutively and prospectively studied: serum samples from 1000 ambulatory patients, 286 serum samples from patients for which anti-CCP was requested and 89 serum samples from patients which had previously given a positive anti-CCP result on Architect® i2000.The frequency of confirmed anti-streptavidin IgG-positive samples detected in this study was 0.6% (8/1375). Anti-CCP IgG was determined on the eight samples with confirmed anti-streptavidin IgG antibodies: with the Cobas® method, seven positive anti-CCP results were observed and five positive anti-CCP results with the Architect® method. No positive anti-CCP IgG results were obtained with the EliA™ method. Rheumatoid factor was negative in these eight samples.Anti-streptavidin IgG antibodies rarely cause false-positive results in some anti-CCP assays. However, despite being an infrequent assay problem, it could possibly lead to diagnostic confusion or even an incorrect diagnosis of rheumatoid arthritis.