The binding of blasticidin S to ribosomes is demonstrated using equilibrium dialysis. There is a single binding site per each ribosome and the association constant is 5×105 M-1. The binding site is localized in the 50S ribosomal subunit. The binding of blasticidin S to ribosomes is inhibited by gougertotin, but not affected by chloramphenicol, lincomycin, erythromycin or puromycin.