We report a simple and reliable method for detection of two or more antigens within tissue sections by indirect immunoenzyme staining using mouse monoclonal antibodies (MAbs). This technique involves treating sections with two 5-min microwave (MW) oven heatings between sequential rounds of three-layer immunoenzyme staining (mouse MAb, goat anti-mouse IgG, and mouse PAP or mouse APAAP) and color development. Discrete staining of cell surface, cytoplasmic, and nuclear antigens was evident within individual cells. This technique has a number of advantages over those currently available. First, MW treatment denatures bound antibody molecules, thereby completely blocking crossreactivity between sequential rounds of staining. This allows the use of primary (and other) antibodies raised in the same species and the use of a sensitive three-layer staining method. Second, antigen retrieval after MW treatment markedly increases the sensitivity of cytoplasmic and nuclear antigen detection. Third, inactivation of peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes prevents inappropriate color development. Finally, this method can be used in both paraformaldehyde-fixed cryostat sections and formalin-fixed paraffin tissue sections. In conclusion, this is a simple, reliable, and sensitive technique that will be useful in many areas of diagnosis and research.