The nucleotide sequences surrounding 1-methyladenylic acid in transfer ribonucleic acid from rat liver and from yeast were investigated by fractionation of pancreatic ribonuclease digests of transfer ribonucleic acid samples. Six 1-methyladenine-containing oligonucleotides were isolated from rat liver transfer ribonucleic acid digests, and the sequences were identified as guanylyl-(3′,5′)-1-methyladenylyl-(3′,5′)-uridylic 3′-acid, adenylyl-(3′,5′)-1-methyladenylyl-(3′, 5′)- uridylic 3′-acid, guanylyl-(3 ′,5 ′)-1-methyladenylyl-(3 ′,-5 ′)-cytidylic 3 ′-acid, guanylyl-(3′,5′)-1-methyladenylyl- (3′,5′)-adenylyl-(3′,5′)-uridylic 3′-acid, adenylyl-(3′,- 5′)-1-methyladenylyl-(3′,5′)-adenylyl-(3′,5′)-uridylic 3′- acid, and guanylyl-(3′,5′)-1-methyladenylyl-(3′,5)-adenylyl-(3′,5′)-cytidylic 3′-acid. The same six oligonucleotides were found in yeast transfer ribonucleic acid. About 30% of yeast transfer ribonucleic acid molecules contain no 1-methyladenine, and the majority of these could be methylated in vitro using a purified transfer ribonucleic acid methylase preparation derived from rat liver. When [14C]S-adenosylmethionine was used as methyl donor for methylation, the radioactive 1-methyladenine so produced was found in the same six oligonucleotides as were found in yeast and liver transfer ribonucleic acid. The basis by which a transfer ribonucleic acid methylase recognized its substrate has been discussed in terms of these results. © 1969, American Chemical Society. All rights reserved.