Deleterious 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions are introduced into nucleic acids by methylating agents. It was recently demonstrated that the E. coli AlkB protein and a human homolog, hABH3, can demethylate these lesions both in DNA and RNA. To elucidate the biological significance of the RNA repair, we have tested whether such repair can rescue the function of chemically methylated RNA. We demonstrate that a methylation-induced block in translation of an mRNA can be readily relieved by treatment with AlkB and hABH3 prior to translation. Furthermore, we show that chemical methylation of tRNAPhe inhibits aminoacylation and translation, but that the inhibition can be reversed by AlkB and hABH3. AlkB-mediated repair of 1-meA in tRNA was also observed in E. coli in vivo. Our data demonstrate that AlkB proteins can mediate functional recovery of RNA exposed to methylation damage, supporting the notion that RNA repair is important.