Virginiamycin S (VS, a type B component of the synergistin group of antibiotics) is fluorescent in solution: the fluorescence intensity is proportional to VS concentration. The intensity of VS fluorescence was found to increase upon addition of 50S ribosomal subunits, and this variation (deltaI 416 nm) to be proportional to the concentration of 50S subunits. This new technique was, then, used to measure the binding reaction of VS to ribosomes. Similar patterns of linkage were obtained for ribosomes and large subunits, whereas very little fixation to 30S particles was detected. The binding reaction was virtually instantaneous at any temperature, and, for saturating VS, was not influenced by Mg++ concentration in the range 1 to 20 mM, nor by the replacement of 100 mM K+ with NH+4. The association constant of VS TO 50S particles was found to be KA=2.5 X 10(6)M-1, and from the Scatchard plot a v value of 0.9 was calculated, which points to a stoichiometric reaction leading to 1 mole VS bound per mole of 50S particles. Upon fixation of virginiamycin M (VM, a type A component of the synergistin group of antibiotics), the delta I of the VS-ribosome complex was increased, and a KA=15 x 10(6)M-1 was recorded for the association constant of VS to 50S particles. Such sixfold increase in the affinity of ribosomes for VS may account for the synergistic effect of the 2 virginiamycin components in sensitive bacteria.