Ribosomes from Escherichia coli were incubated with sulfhydryl reagents and then after removal of the excess reagent assayed for polyuridylic acid dependent polyphenylalanine synthesis in the absence of both sulfhydryl reagents and thiols. Incubation with N-ethylmaleimide, dithiobis-(2-nitrobenzoic acid) and p-chloromercuribenzoic acid resulted in loss of activity between 30 and 70% in different ribosome preparations. Similar inactivation was observed in the polylysine assay system and in polyuridylic acid directed leucine incorporation (miscoding). Inactivation by dithiobis-(2-nitrobenzoic acid) was reversed by incubating the ribosomes with a thiol. The extent of inactivation was independent of whether the ribosomes were treated with the sulfhydryl reagent in the associated (70 S) or dissociated (50 S + 30 S) form. The sedimentation coefficients of the treated ribosomes and of the subunits prepared from them were not altered. The treated ribosomes were in the associated (70 S) form under the conditions of polyphenylalanine synthesis. No inhibition of the binding of polyuridylie acid was found. Inactivation occurred when the molar ratio of sulfhydryl reagent to 70 S ribosome was only 2 or 3, although the calculated half cystine content of the 70 S ribosome is 45 residues. The 30 S ribosome was the principle site of inhibition. The results indicate that at least some ribosomal half cystine is present as cysteine and that ribosomal SH groups, as well as supernatant transfer factor SH groups, are important in protein synthesis.