According to the allosteric three-site model for the ribosomal elongation cycle, the reactions from the pre- to the post-translocational state and vice versa represent allosteric transitions which are catalyzed by elongation factor (EF)-G and EF-Tu, respectively. It has been shown recently that the non-related antibiotics thiostrepton and viomycin inhibit protein biosynthesis via a surprisingly similar mechanism. Both drugs primarily block the allosteric transitions in either direction (Hausner et al. (1988) J. Biol. Chem. 263, 13103-13111). Here we show that the secondary effects of these antibiotics differ strikingly. When the P site of poly(U) programmed ribosomes is quantitatively filled with AcPhe-tRNA, thiostrepton stimulates the rate of the formation of AcPhe-puromycin 2-fold, whereas viomycin inhibits the puromycin reaction (up to 75% inhibition). The thiostrepton-dependent stimulation is only observed when the drug is given before the P site is occupied; when thiostrepton is added after pre-filling the P site, the peptidyltransferase activity is not affected, in contrast to the translocation reaction, which is blocked irrespective of whether the drug is administered before or after tRNA is bound. The effects of both drugs became distinctly more pronounced when the P sites were saturated with AcPhe-tRNA as compared to half-saturated ribosomes. We conclude that roughly one half of the ribosomes, which first bind AcPhe-tRNA to the P site, carry this ligand in a different orientation to that of the second half of the ribosome population. These two populations probably reflect the P site in the pre- and post-translocational state, respectively.