Kethoxal reacts with 30S ribosomal subunits to give totally inactive particles, as measured by in vitro protein synthesis. It is postulated that functional modification occurs at the binding site for transfer RNA since ( a ) loss of specific binding of transfer RNA, but not binding of messenger RNA, is simultaneous with loss of protein synthesis, and ( b ) loss of activity is inhibited by bound transfer RNA. By means of in vitro reconstitution and labeling techniques, it is found that loss of transfer RNA-binding activity is correlated with the modification of six or seven guanine residues in 16S RNA.