The major advantages of the horseradish peroxidase chemiluminescence (HRP-CL) immunodetection method in Western blot analysis are its high sensitivity, nonradioactive detection, economy of the primary antibody, and speed of detecting the signal. However, we observed a strong and reproducible signal that was detected regardless of the primary antibody and of the cell type used. This signal, present at 56-54 kilodaltons (kDa), is generated in absence of any primary antibody and seems to be an intrinsic reaction of the HRP-labeled second antibody anti-immunoglobulin with an unidentified cellular protein. The use of dry milk throughout all the steps of the procedure abolishes this signal. For those interested in one of the numerous proteins migrating at or close to 56-54 kDa, the question therefore arises as to which signals generated by the HRP-CL in this region are bona fide and which are non grata pseudo signals.