The mode of action of the antiviral drug 5-(3,4-dichlorophenyl) methylhydantoin (hydantoin) was studied in a cell-free system allowing de novo synthesis of poliovirus. This cell-free system, which is programmed with viral RNA, is suitable for the study of the late stages of poliovirus replication and, thus, for a study of antiviral compounds acting on these late stages. It was shown that, apart from the known inhibition of the assembly of poliovirus, hydantoin also blocks post-synthetic cleavages of poliovirus proteins. Our data demonstrate that the cell-free system is a sensitive tool to study the mode of action of antiviral compounds.