Rapidly progressive glomerulonephritis (RPGN) is characterized by rapid and progressive loss of renal function and the presence of crescentic glomerulonephritis (CGN). Early diagnosis and appropriate treatment is mandatory to prevent death and/or renal failure. We have evaluated an ANCA-GBM dot-blot diagnostic test in terms of sensitivity, specificity, and inter-observer effect in consecutive patients with RPGN ( n = 82). Control sera ( n = 34) included healthy and relevant disease controls. Dot-blots were independently evaluated by nine observers. Proteinase 3 (PR3)-ANCA, myeloperoxidase (MPO)-ANCA, and both were detected by ELISA in 36, 32, and 3 samples of 71 patients with pauci-immune CGN, respectively. Two additional samples were ANCA negative. The dot-blot revealed a sensitivity of 92-95% for PR3-ANCA and 80-86% for MPO-ANCA. The specificity of the dot-blot for PR3- and MPO-ANCA was 100%. In the patients with anti-GBM nephritis ( n = 9) anti-GBM was detected by both ELISA and dot-blot (sensitivity: 100%). The specificity of the anti-GBM dot-blot was 91-94%. However, the inter-observer effect was relatively high for detection of anti-GBM antibodies (24%). In conclusion, the ANCA-GBM dot-blot is a useful screening tool in situations where conventional ANCA testing is not readily available with excellent performance for PR3-ANCA detection, but less optimal sensitivity for MPO-ANCA and specificity for anti-GBM detection. Therefore, it is recommended to include the following advises in the report to the physicians: 1) patients with a high clinical suspicion for MPO-ANCA-associated RPGN and negative dot-blot must have conventional analysis for MPO-ANCA, and 2) negative anti-GBM dot-blot makes anti-GBM disease very unlikely, but positive samples should be confirmed by conventional anti-GBM tests.