Monoclonal antibodies of known specificity, and target cells of known antigen expression, were used to evaluate the specificity of the immunoblot assay against complex extracts. The antibody panel included 10 previously characterized monoclonals to antigens of known structure: HLA-A,B,C; Ia; and actin. In addition, a commercial reagent was shown to react with HLA chains in immunoblots, and not to cross-react with actin. Target cells known to have strong, weak, or negative expression of the antigens were examined. In all cases, the results were in complete accord with the established properties of the antibodies, antigens, and target cells. For example, HLA bands were detected in lymphoid cells with strong HLA-A,B,C expression, and also in a neuronal cell line with 0.5% of the lymphoid activity. Cytoskeletal cross-reactions, or other unexpected specific bands, were not observed. This work lays a foundation for interpreting immunoblot analyses of monoclonal antibodies to newly defined proteins of complex tissue.