Five antibiotics (puromycin, erythromycin, lincomycin, sparsomycin, and virginiamycin M1) that bind specifically to the 50 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation of fluorescence from probes attached to the alpha-amino group of Phe-tRNA. Derivatives of three probes with differing chemical characteristics and environmental sensitivities were used: a coumarin, an aminonaphthalenesulfonate, and a pyrene. The effects of the antibiotics on the fluorescence of labeled AcylPhe-tRNAs in the two states, while generally qualitatively similar, are nonetheless quantitatively distinct, as are the calculated binding constants for the antibiotics. Puromycin, as reported earlier, binds to both the puromycin-reactive and -unreactive states, but its dissociation constant is higher for the latter state. Erythromycin binds tightly to ribosomes bearing labeled AcylPhe-tRNA in either the puromycin-reactive or -unreactive state. Its effect on the fluorescence of the labeled tRNA is very similar in the two states, except with the pyrene probe, where it has a larger effect in the puromycin-reactive state. Lincomycin and sparsomycin bind to both ribosomal states, but both bind more tightly to the puromycin-reactive state, the extent of the difference varying with the identity of the fluorescent probe. Virginiamycin M1 binds to ribosomes with AcylPhe-tRNA in the puromycin-reactive site, but its binding could not be detected to ribosomes with AcylPhe-tRNA in the puromycin-unreactive site.