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Antibiotics that target the eukaryotic ribosome represent potential antitumor and anti-inflammatory drugs. For many of them, the functional details of their activity remain incompletely understood. Using toe-printing technique, we analyzed effects of several translation inhibitors in mammalian cell extract. When pre-incubated with the lysate before mRNA addition, both aminoglycoside antibiotics and translocation inhibitors predictably stopped elongating ribosome at the very beginning of the coding region. In contrast, antibiotics that affected the peptidyl transferase center (PTC) of the 60S subunit demonstrated complex patterns of toe-print signals along the mRNA. We investigated this phenomenon in detail in a case of antitumor drug omacetaxine mepesuccinate (homoharringtonine) and its derivative harringtonine, which had been used recently in ribosome profiling studies. We showed that both drugs specifically halted elongating ribosomes at Lys, Arg or Tyr codons positioned in the P-site. Statistical analysis of ribosome profiling data generally confirmed this conclusion in a transcriptome-wide scale. Molecular modeling suggested that such specificity was dictated by additional contacts of the antibiotic side chain with peptidyl moiety of the P-tRNA. We unexpectedly found the same toe-print pattern for a chemically distinct trichothecene antibiotic, T-2 toxin, while another inhibitor of this group, diacetoxyscirpenol, produced a completely distinct picture of ribosome stops. Furthermore, the Fleeting RNA Transfection (FLERT) of living mammalian cells revealed an intriguing specificity of the PTC inhibitors toward reporter mRNAs with different 5’ untranslated regions. Our data suggests that the mechanism of inhibition of the protein synthesis by PTC inhibitors is more complex than it was originally proposed. The work was supported by RFBR (grant no. 16-04-01271) and the grant of the Russian Federation government N2016-220-05-308 (14.W03.31.0012).