2'-O-methylation of eukaryotic ribosomal RNAs occurs in the cell nucleoli. At least 100 modification sites that are highly conserved among vertebrate rRNAs have been mapped. However, in part because of the insensitivity of current approaches, there are 2'-O-methylated sites that remain unidentified. We have developed an extremely sensitive method for detecting 2'-O-methylated residues that are predicted within a long RNA molecule. Utilizing RNase H cleavage directed by a 2'-O-methyl RNA-DNA chimeric oligonucleotide, this method has allowed identification of two methylated nucleotides, G1448 in Xenopus 18S rRNA and A394 in Xenopus 28S rRNA. The latter (A394 in 28S) had not been detected before. We have confirmed that the methylation at G1448 in 18S is dependent upon Xenopus U25 snoRNA and have demonstrated that the methylation at A394 in 28S requires U26 snoRNA. One advantage of this technique is that it can examine specific rRNA and precursor molecules. We show that about 30% of the 40S pre-rRNA has been methylated at these two sites and their methylation is complete at the stage of 20S (immediate precursor to 18S) and 32S (immediate precursor to 28S). We also show that methylation at these two sites is not essential for rRNA transport from the nucleus to the cytoplasm.