0-Methylhydroxylamine (methoxyamine) was used for selective modification of cytosine residues in Escherichia coli 16-S rRNA. It was shown that cytosines accessible for methoxyamination are randomly distributed along the 16-S rRNA chain.Preparations of methoxyaminated 16-S rRNA, containing 2 -130 modified cytosines/chain, still retained the ability to bind 30-S proteins, but the physical assembly of reconstituted particles was incorrect. The protein compositions of the reconstituted and native particles did not differ qualitatively from each other. However, the amount of protein in reconstituted particles decreased with an increasing number of methoxyaminated cytosines in 16-S rRNA.The particles obtained sedimented slower than native 30-S subunits, lost their ability to associate with 50-S ribosomes and to bind native phage f2 RNA. In contrast, modification of 16-S rRNA did not affect binding of poly(U) by reconstituted particles.Chemical modification of RNA is one of the ways to study the relationship between its structure and function. In the previous experiments with ribosomal RNA, however, unspecific reagents, which alter different bases were used. Modification of 16-S rRNA by nitrous acid led to deamination of adenine, guanine and cytosine and blocked the incorporation of phenylalanine in the presence of reconstituted particles containing this RNA [l]. Similar results were obtained by using monoperphthalic acid, which alters adenine and cytosine [2].In our experiments 0-methylhydroxylamine (methoxyamine) was employed as a reagent for selective modification of cytosines in the single-stranded regions of E. coli 16-S rRNA. The effects of this alteration on the reconstitution of 30-S particles, the reassociation of the resulting 30-S with 50-S subunits and binding of poly(U) and f2 RNA to modified ribosomes are described. Attempts were also undertaken to localize methoxyaminated cytosines in the 16-S rRNA molecule.Recent ideas about the participation of cytosines in 16-S rRNA in binding of mRNA to 30-S subunits [3,4] and the involvement of ribosomal RNA-s in the association of subunits [5] made these experiments additionally attractive.