It was previously shown that virginiamycin M produces in vivo an accumulation of pressure-sensitive (60 S) ribosomes, and in vitro an inactivation of the donor and acceptor sites of peptidyl transferase. The latter action, however, is expected to cause the accumulation in vivo of ribosome complexes carrying acylated tRNA species: such complexes are usually endowed with pressure resistance. However, present data indicate that poly(U).ribosome complexes carrying Phe-tRNA, Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at either the A or the P site become pressure-sensitive after exposure to virginiamycin M in vitro. It is known also that uncoupled EF-G GTPase is stimulated by P-site-bound unacylated tRNA, not by the acylated species. Our data show, however, a stimulation of EF-G GTPase, when ribosomal complexes carrying Ac-Phe-tRNA or Ac-Phe-Phe-tRNA at the P site are incubated with virginiamycin M. The interpretation proposed to account for all these findings is that complexes carrying A- and P-site-bound aminoacyl-tRNA derivatives, which undergo a stable interaction with the peptidyl transferase, are endowed with ultracentrifugal stability, whereas complexes with unacylated tRNA (which does not interact with the enzyme) are pressure-sensitive. By inactivating the donor and acceptor sites of peptidyltransferase, virginiamycin M causes aminoacyl-tRNA.ribosome complexes to mimic tRNA.ribosome complexes in their pressure-lability and competence in EF-G GTPase stimulation. This interpretation is supported by the finding that the ribosome-promoted protection of aminoacyl-tRNA against spontaneous hydrolysis is suppressed by virginiamycin M.