In this study, we investigated the reaction of the osmium tetroxide-bipyridine complex with pyrimidines in RNA. This reagent, which reacts with the diastereotopic 5-6 double bond, thus leading to the formation of two diastereomers, was used in the past to label thymidine and 5-methylcytosine in DNA. In light of the growing interest in post-transcriptional RNA modifications, we addressed the question of whether this reagent could be used for labeling of the naturally occurring RNA modifications 5-methylcytosine and 5-methyluridine. On nucleoside level, 5-methylcytosine and 5-methyluridine revealed a 5- and 12-fold preference, respectively, over their nonmethylated equivalents. Performing the reaction on an RNA level, we could show that the steric environment of a pentanucleotide has a major detrimental impact on the reaction rate of osmylation. Interestingly, this drop in reactivity was due to a dramatic change in diastereoselectivity, which in turn resulted from impediment of the preferred attack via the si side. Thus, while on the nucleoside level, the absolute configuration of the major product of osmylation of 5-methyluridine was (5R,6S)-5-methyluridine glycol-dioxoosmium-bipyridine, reaction with an RNA pentanucleotide afforded the corresponding (5S,6R)-diastereomer as the major product. The change in diastereoselectivity lead to an almost complete loss of selectivity toward 5-methylcytosine in a pentanucleotide context, while 5-methyluridine remained about 8 times more reactive than the canonical pyrimidines. On the basis of these findings, we evaluate the usefulness of osmium tetroxide-bipyridine as a potential label for the 5-methyluridine modification in transcriptome-wide studies.