Virginiamycin S (VS) binds to the 50 S ribosomal subunits (KaVS = 2.5 X 10(6) M-1); the affinity of ribosomes for VS undergoes a 6-fold increase (KaVS = 15 X 10(6) M-1) in the presence of virginiamycin M (VM). In the present work, the action of inorganic ions and pH on the binding reaction of VS to ribosomes was analyzed by a spectrofluorimetric technique, in the presence and in the absence of VM. Preliminary to this study, the interaction of ions with free VS was also explored. In aqueous solution and in the absence of VM, chelation by VS of K+, NH4+, Na+, Mg2+ and Ca2+ was observed. The binding of Mg2+ was strongly influenced by monovalent ions and pH between 7 and 8: the association constants for the VS:Mg2+ complex were 12.5 M-1 (+NH4+) and 45 M-1 (-NH4+). Binding of VS to ribosomes occurred as well in the presence of NH4+ as with K+, but was suppressed upon replacement of these ions by Na+. The association constant of the VS binding reaction to ribosomes was strongly enhanced by VM in the presence of either NH4+ or K+ but, while a plateau value was observed over the entire range of NH4+ concentrations, 100-fold higher values were obtained at low (25 mM) K+. This VM-induced increase of ribosome affinity for VS (KaVS) was dependent also on Mg2+, which displayed a higher synergistic action with K+ than with NH4+ (the highest KaVS values were recorded for 5 mM Mg2+ and 100 mM K+). In this reaction, Mg2+ could be replaced by Ca2+, whereas spermidine was ineffective. Also, the VM-promoted enhancement of the KaVS value increased with the pH. In conclusion, the two ribosomal functions analyzed in the present work, VS-binding to ribosome and VM-promoted enhancement of ribosome affinity for VS, rely on the concentration of monovalent and bivalent ions and on the pH. Both functions require either NH4+ or K+, and either Mg2+ or Ca2+ (the elements of each couple are not truly equivalent), from which ribosome conformation depends.