Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes. Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site. Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry. Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can bc exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative.