The antigenic structure of tobacco mosaic virus has been analysed by measuring the ability of nine monoclonal antibodies to distinguish between wild-type virus and 13 mutants showing single and double amino acid substitutions in the coat protein. Although the majority of antibodies detected those substitutions that were located at the outer surface of the virion, some of them also recognized conformation alterations induced by exchanges occurring deep inside the subunit. In the case of five mutants, the antibody reactivity was reduced compared with wild-type virus, while in the case of three others, it was significantly higher. Each monoclonal antibody possessed a unique discrimination pattern with respect to the different substitutions. The simultaneous presence of two exchanges led to the complete disappearance of any binding with six of the nine antibodies and to reduced binding with three others. The superior discriminatory capacity of monoclonal antibodies compared with polyclonal antisera was demonstrated by the fact that three exchanges not detected with antisera were found to alter the antigenicity when tested with monoclonal antibodies.