Several antibiotics including anthelmycin, blasticidin S, destomycin A, gougerotin, hygromycin B and edeine complex, known to powerfully block translation in cell-free systems, did neither inhibit protein synthesis in intact mouse L and 3T6 cells, nor in hamster BHK 21 cells, due to failure to cross the cell plasma membrane. However, after viral infection, these antibiotics exhibited a marked blockade of translation, that is related to the permeability changes induced by viral infection. The inhibition of protein synthesis by hygromycin B in virus-infected cells was studied over the time course of infection, both in encephalomyocarditis virus-infected mouse L cells and in Semliki forest virus-infected hamster BHK cells. We have observed that the entry of hygromycin B into virus-infected cells parallels the inhibition of cellular protein synthesis, i.e., the cells became permeable to this antibiotic at the time the shut-off of host translation occurred. A marked inhibition of picornavirus RNA synthesis by hygromycin B was also noticed, likely as a consequence of the inhibition of the viral replicase synthesis. Finally, a reduction in the virus yield by treatment of virus-infected cells with several antibiotics is also described. All these observations are considered in the context of the interference of viral infection with cellular functions and the potential use of inhibitors non-permeable to normal cells as antiviral agents.