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Cycloheximide is a well-known antibiotic of the glutarimide family with a high specificity towards the eukaryotic ribosome. The drug inhibits translocation by interfering with CCA-end of deacylated tRNA in the E-site of the large ribosomal subunit. Cycloheximide is widely used to inhibit protein synthesis in eukaryotic cells and to freeze translating ribosomes for polysome profile analysis and ribosome profiling studies. Here we applied a mammalian cell-free system with continuous monitoring of luciferase mRNA translation in a standard plate reader to analyze cycloheximide activity in vitro. We detected a pronounced delay in product appearance even at low drug concentrations that do not significantly affect overall translation efficiency. We concluded that cycloheximide molecules dynamically interact with roughly all ribosomes in the system, thus evenly distributing between polysomes and leading to a general slowing of ribosome progression. This mode of action fundamentally differs from that of other elongation inhibitors (i.e. emetine, pactamycin, amicoumacin A, anisomycin, harringtonine and others) which, taken at low concentrations, block only a portion of polysomes in the system and do not cause the delay. The two activity modes of antibiotics are discussed in a context of their differential effects on living cells, where short-term translation inhibition by cycloheximide is fully reversible while a similar treatment with other antibiotics often triggers a ribotoxic stress response and apoptosis. The same peculiarity of cycloheximide action could also explain the well-known artifacts found in ribosome profiling data obtained with this drug. Finally, we used toe-printing technique to clearly demonstrate that cycloheximide arrests the elongating ribosome by inhibition of deacylated tRNA movement from the P- to the E-site occupied by the drug and not by trapping the translocated tRNA in the latter, as suggested earlier.