Simple two-dimensional thin-layer chromatography was found to be useful for the separation of sugar methylated dinucleotides in RNA. Of the 16 possible sequences of the type Nm-Np, 15 were separated and all the sequences were determined. In a mouse hepatoma, MH 134, the levels of the sugar methylation in the 18S and 28S RNA molecules were 17-18 and 11-12 per 1000 nucleotides, respectively. Thus, 18s RNA contained approximately 35 2'-O-methylated dinucleotides and 28S RNA approximately 60 2'-O-methylated dinucleotides. The pattern of distribution was also distinct between these two molecules. Two 2'-O-methylated trinucleotides were identified in the 28S RNA with the sequences Um-Gm-Up and Um-Gm-psip. A unique 2'-O-methylated tetranucleotide was present also in the 28S RNA, the sequence of which was Am-Gm-Cm-Ap. The 5'-terminal nucleotides of both 18S and 28S RNA were obtained as nucleoside 3',5'-diphosphates (pNp) in the trinucleotide fraction of the RNase T2 digest. The 5'-termimi of 18S and 28S RNA were pUp and pCp, respectively, and found to be almost homogeneous.