Technical Abstract: Infectious agents or their constituent parts (antigens or nucleic acids) can be detected in fresh, frozen, or fixed tissue using a variety of direct or indirect assays. The assays can be modified to yield the greatest sensitivity and specificity but in most cases a particular methodology is chosen based on the nature of the sample, availability of equipment, quality of reagents, time, cost, and technical expertise. Whole virions or bacteria can be visualized directly using electron microscopy. Although this procedure can be performed quickly, it is insensitive and requires specialized equipment and technical support. Some tests such as agglutination reactions or immunodiffusion assays are simple and inexpensive but they can only be used to identify a particular etiologic agent possessing agglutinative or soluble antigens, respectively. The more cumbersome techniques such as antigen-capture enzyme-linked immunosorbent assay, immunohistochemical staining, or in situ hybridization offer increased sensitivity and specificity but may require better quality reagents, additional pretreatment procedural steps prior to testing, and a higher level of technical training. To optimize antigen detection systems for diagnostic and research purposes it is as important to become familiar with the limitations of the assays as it is to understand the pathogenesis of the suspected etiologic agent in selecting the timing and type of tissue samples for testing.