The affinity of ribosomes for VS (virginiamycin S, a type B synergimycin) is known to be increased by VM (virginiamycin M, a type A synergimycin). Erythromycin, a macrolide, displaces ribosome-bound VS in the absence of VM, but is ineffective in its presence. In the present work, the ability of spiramycin and tylosin (macrolide subgroups) derivatives to displace ribosome-bound VS, in the presence and in the absence of VM, has been explored. All macrolides with in-vitro activity displaced ribosome-bound VS: the displacement curves produced by tylosin and spiramycin derivatives virtually overlapped. When VM was added to these systems, displaced VS became readily attached to ribosomes in the case of erythromycin, did not bind appreciably within 20 min incubation in the presence of tylosin, and underwent a slow binding in the case of an N-substituted tylosin. The 16-membered macrolides (leucomycin, spiramycin and tylosin subgroups) can therefore be distinguished from the 14-membered macrolides (erythromycin subgroup) by the antagonistic effect displayed toward VM.