Oxazolidinones are antibacterial agents that act primarily against gram-positive bacteria by inhibiting protein synthesis. The binding of oxazolidinones to 70S ribosomes from Escherichia coli was studied by both UV-induced cross-linking using an azido derivative of oxazolidinone and chemical footprinting using dimethyl sulphate. Oxazolidinone binding sites were found on both 30S and 50S subunits, rRNA being the only target. On 16S rRNA, an oxazolidinone footprint was found at A864 in the central domain. 23S rRNA residues involved in oxazolidinone binding were U2113, A2114, U2118, A2119, and C2153, all in domain V. This region is close to the binding site of protein L1 and of the 3′ end of tRNA in the E site. The mechanism of action of oxazolidinones in vitro was examined in a purified translation system from E. coli using natural mRNA. The rate of elongation reaction of translation was decreased, most probably because of an inhibition of tRNA translocation, and the length of nascent peptide chains was strongly reduced. Both binding sites and mode of action of oxazolidinones are unique among the antibiotics known to act on the ribosome.