Abstract An immunoblotting method is described that permits sequential detection of multiple antigens on a single protein blot without stripping off prior antibodies. The procedure utilizes horseradish peroxidase (HRPase)- based detection with a chemiluminescent substrate. After detection by enhanced chemiluminescence (ECL) with exposure to X-ray film, the antigen–antibody complexes on the blot are reacted with a chromogenic substrate (either 3,3′-diaminobenzidine [DAB] or SG [Vector Labs, Inc.]) which renders the antigen–antibody–HRPase complexes unreactive in subsequent reprobings of the same membrane with additional antibodies using the same detection method. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple sequential reprobings of the same membrane with different primary antibodies (≥12) and retention of strong signal intensities for all antibody probings. A variation of the method (the “rainbow Western”) is described in which four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same blot. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples that can be obtained are limiting or only available on a one-time basis.