The M and S components of virginiamycin (VM and VS) inhibit protein synthesis in bacteria--reversibly when a single component is present and irreversibly when both are present. In cell-free systems, each factor binds to the large ribosomal subunit, and the affinity of ribosomes for VS is enhanced in the presence of VM. The present work shows that the action of VM (a 500-dalton modified depsipeptide) in vivo and in vitro persists upon its removal. The in vivo demonstration is based on the loss of viability of uninfected bacteria, and on the irreversible inactivation of virus-infected cells, that are caused by a sequential incubation with VM and VS (the inhibitory action of either component alone is reversible). In vitro, the binding of labeled VM to ribosomes, followed by its detachment, yields particles unable to perform poly(U)-directed polyphenylalanine synthesis. Also, the association constant for the binding of VS to these particles is equal to that of particles incubated with a mixture of VM and VS. Our findings indicate that VM action is catalytic rather than stoichiometric, and suggest the occurrence of two states of the large ribosomal subunit, a situation leading to a complex equilibrium with multiple transitional steps in the presence of virginiamycin.