The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L-[methyl-³H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT)-cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl-labeled nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE-cellulose (borate) chromatography, which separates 2′-O-methylnucleosides from normal and base-methylated nucleosides, about 50% of the radioactivity was recovered in the 2′-O-methylnucleoside fraction and 50% in the base-methylnucleoside fraction. High-speed liquid chromatography (Aminex A-5) of the 2′-O-methylnucleoside fraction produced four peaks coincident with the four 2′-O-methylnucleoside standards. Analysis of the base-methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base-methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N⁶-methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences.