The inhibition by thiostrepton of the initiation of protein synthesis is exerted at a different level from the inhibition of reactions mediated by EF-Tu and EF-G in the elongation of protein synthesis. The presence of thiostrepton on the 50-S subunit completely prevents the binding of the EF-Tu - GTP - aa-tRNA complex and EF-G - GTP complex to the 70-S ribosome, resulting in cessation of protein synthesis at a concentration of 1 muM thiostrepton. On the other hand, during initiation thiostrepton impairs the coupling of the 50-S subunit with the 30-S initiation complex, indirectly causing inhibition of IF-2-dependent reactions. Impairment of the coupling is strongly influenced by the conditions of incubation. Since formation of formylmethionylpuromycin and the IF-2-dependent GTP hydrolysis are inhibited to the same extent and recycling of IF-2 can take place in the presence of thiostrepton, we conclude that the basic mechanism of inhibition of initiation differs from that of inhibition of elongation.