The modes of action of antirhinovirus agents and mechanisms of resistance to the agents are compared. Chalcone Ro 09-0410, 4\',6-dichloroflavan (DCF) and RMI-15,731, which were active against rhinoviruses, inactivated the virus directly. Inactivation was associated with the binding of the agents to the virus particles, since the infectivity, reduced by exposure to the compounds, was restored to the original level by extraction of the agents with chloroform. The binding of [3H]chalcone to rhinovirus type 2 was inhibited by any of the three unlabelled agents. Furthermore, virus sublines selected for resistance to both dichloroflavan and RMI-15,731 showed cross-resistance to chalcone and vice versa. These findings indicate that the three agents exert their activities through the same mode of action (namely binding to or interaction with a specific site on the viral capsid protein) and that the binding or interaction sites for these three agents are either the same or very close to each other. Since the sublines resistant to chalcone and to RMI-15,731 have little or no capability to bind to chalcone, the resistance to these agents is conferred by changes in the viral capsid protein. On the other hand, flavone Ro 09-0179 and enviroxime, which were active widely against picornaviruses, had no ability to inactivate the virus directly, and their antiviral activity was not associated with the capsid protein.