There is an average of about one residue of N4,O2′-dimethylcytidine per polynucleotide chain in Escherichia coli 16-S rRNA. This compound has a methyl substituent in both base and sugar, and has, as yet, been detected only in the slower sedimenting component of the bacterial rRNA. This rare nucleoside has been prepared from E. coli rRNA in sufficient quantity to allow for a detailed structural analysis by well-established degradative and spectrophotometric procedures. The base and sugar have been characterized as N4-methylcytosine and O2′-methylribose, respectively. The sugar has a furanosyl ring structure and is attached in the β-configuration at N-1 of N4-methylcytosine. Allied studies have shown that all constituent nucleosides in the alkali-stable, O2′-methylribose-containing dinucleotide sequences of wheat embryo rRNA exist in the β-configuration. © 1968.