Indolmycin specifically prevents the formation of tryptophanyl-tRNA in a prokaryotic system in vifvo using Eschevichia coli enzymes. However, the drug has little effect in an eukaryotic system in v i m (rat liver enzymes). Analysis of the type of inhibition revealed that indolmycin competes with tryptophan as a pure competitive inhibitor of prokaryotic tryptophanyl-tRNA ligase.Most of the antibiotics inhibiting protein synthesis interfere directly with prokaryotic ribosomal functions. A small number of antibiotics act as inhibitors of steps taking place prior to translation (for review see [l]). For example, the drug borrelidin prevents the formation of threonyl-tRNA in some bacterial speciesIn this paper we show that indolmycin specifically inhibits the tryptophanyl-tRNA ligase from Escherichia coli whereas the corresponding ligase from rat liver is affected much less.PI. MATERIALS AND METHODS Muter ialsATP and tRNA from E. coli and calf liver were obtained from Boehringer, Mannheim ; the radioactive amino acids were obtained from New England Nuclear; Instagel scintillation liquid from Packard ; the filters from Sartorius ; the total synthesis of indolmycin followed described procedures [3 -61; all other chemicals were from Merck, Darmstadt. The 300000xg supernatant from rat liver was a generous gift from Dr F. Grummt