The antibiotic, thiostrepton, binds to 23S ribosomal RNA from E coli with a dissociation constant (KD) of 2.4 x 10(-7) M. The specificity of the interaction was established using 16S rRNA and modified or mutationally-altered 23S rRNA. Thus, no binding was detected with rRNA from the 30S subunit nor with rRNA modified in vitro by the thiostrepton resistance methylase. Mutant 23S rRNA, altered at residue 1067 in each of the 3 possible ways, showed reduced binding affinity for thiostrepton. The KD for the G mutation was 3.5 x 10(-6) M; for the C mutation, 2.4 x 10(-5) M; and for the U mutation, 4.8 x 10(-5) M. This reduction in drug binding is compatible with functional analyses; the C or U mutation results in ribosomal particles which are poorly inhibited by the drug compared with wild-type, whereas the G mutation results in an intermediate response to the drug in protein synthesis. The smallest 23S rRNA fragment used here that was capable of binding thiostrepton, in a nitrocellulose filter binding assay, comprised residues 1052-1112 and the dissociation constant was 3.0 x 10(-7) M, ie virtually indistinguishable from that with intact 23S RNA. However, the drug was incapable of binding to the 5'-moiety of this fragment (ie residues 1052-1084) or to an RNA transcript complementary to 1052-1112.