Oxazolidinones are a promising new class of synthetic antibiotics active against multidrug-resistant Gram-positive bacteria. To elucidate their mode of action, the effect of DuP 721 on individual steps of protein translation was studied. The drug does not interfere with translation initiation at the stage of mRNA binding or formation of 30S pre-initiation complexes. However, it inhibits the puromycin-mediated release of [35S]formyl-methionine from 70S initiation complexes in a dose-dependent manner. Inhibition involves binding of the oxazolidinone to the large ribosomal subunit and is twice as high with 50S subunits from Gram-positive as with those from Gram-negative bacteria.