Since its introduction in 1975 the methodology of Kohler and Milstein (1) for production of monoclonal antibody (MAb) from hybridoma cells has been widely used to provide antibodies with a defined specificity. One characteristic feature of this technology is that impure antigens can be used to produce monospecific antibodies that can be utilized to study the functional domains of protein molecules. In this chapter, the use of limited vs complete proteolytic digestion experiments to define the epitope on the antigen recognized by a given MAb is outlined. We describe our studies (2) with the transducin (G_t) α-subunit in which proteolytic digestion, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and Edman degradation were used to determine the sequence of the fragments recognized by the MAb 4A. Therefore, reaction times and reagents presented in this chapter may require some modification when a different protein antigen is under investigation.