The accuracy of detection and identification of low-concentration M proteins using capillary zone electrophoresis/immunosubtraction (CZE/IS) plots was systematically studied. To this end, 49 CZE plots (protein spectra; session I) and CZE/IS plots (spectra with corresponding immunosubtraction plots; session II) were judged by participants from 12 Dutch laboratories. Plots were based on mixed sera with a low concentration (0.4, 1 or 4 g/L) of one M protein, as well as normal sera, sera with an oligoclonal or polyclonal increase in immunoglobulins, and sera containing free monoclonal light chains. At 0.4 g/L, nine out of 12 (9/12) different M proteins were hard to detect. At 1 g/L, 6/12 M proteins were detected by > 85% of the participants. At 4 g/L, 10/12 M proteins were detected by all participants. Normal spectra were always judged correctly. At 1 g/L and 4 g/L, detected M proteins were identified correctly by 92% of the participants, versus only 63% at 0.4 g/L. The detection accuracy was only slightly (non-significantly) improved by the application of IS plots. Our data indicate that CZE/IS is applicable for most M proteins at concentrations > or = 4 g/L.