FTO, a protein associated with obesity, efficiently demethylates N6-methyladenosine (m6A) in RNA. In vitro, FTO shows high activity in demethylating m6A in RNA and DNA. Knockdown of FTO in human cells increases m6A levels in mRNA, while overexpression decreases them. FTO partially colocalizes with nuclear speckles, suggesting m6A in nuclear RNA is a physiological substrate of FTO. FTO belongs to the AlkB family of dioxygenases, which also include ABH1-ABH8. These proteins oxidatively demethylate modified DNA and RNA bases. FTO has been shown to demethylate m3T and m3U in ssDNA and ssRNA, but its activity is lower than other AlkB family proteins. The crystal structure of FTO confirms its preference for single-stranded nucleic acids. m6A is the most abundant methylated base in mRNA, and its role in mRNA processing and export is unclear. FTO demethylates m6A in ssDNA and ssRNA, converting it to adenosine. In vitro, FTO shows a strong preference for m6A over m3U. In vivo, FTO knockdown increases m6A levels in mRNA, while overexpression decreases them. FTO is localized to cell nuclei and partially colocalizes with nuclear speckles. FTO activity is affected by pH, with highest activity at pH 6.0. FTO knockdown reduces MT-A70 expression, a subunit of m6A methyltransferase, indicating that the increase in m6A is due to FTO depletion, not increased methyltransferase activity. FTO partially colocalizes with nuclear speckles, supporting the idea that m6A in nuclear RNA is a substrate of FTO. The enzymatic alteration of m6A may be linked to processing of recently transcribed mRNA. FTO's function in demethylating m6A in mRNA suggests a novel, reversible regulatory mechanism in mammalian cells. The discovery of FTO-mediated oxidative demethylation of m6A in nuclear RNA may lead to further investigations on biological regulation based on reversible chemical modification of RNA.FTO, a protein associated with obesity, efficiently demethylates N6-methyladenosine (m6A) in RNA. In vitro, FTO shows high activity in demethylating m6A in RNA and DNA. Knockdown of FTO in human cells increases m6A levels in mRNA, while overexpression decreases them. FTO partially colocalizes with nuclear speckles, suggesting m6A in nuclear RNA is a physiological substrate of FTO. FTO belongs to the AlkB family of dioxygenases, which also include ABH1-ABH8. These proteins oxidatively demethylate modified DNA and RNA bases. FTO has been shown to demethylate m3T and m3U in ssDNA and ssRNA, but its activity is lower than other AlkB family proteins. The crystal structure of FTO confirms its preference for single-stranded nucleic acids. m6A is the most abundant methylated base in mRNA, and its role in mRNA processing and export is unclear. FTO demethylates m6A in ssDNA and ssRNA, converting it to adenosine. In vitro, FTO shows a strong preference for m6A over m3U. In vivo, FTO knockdown increases m6A levels in mRNA, while overexpression decreases them. FTO is localized to cell nuclei and partially colocalizes with nuclear speckles. FTO activity is affected by pH, with highest activity at pH 6.0. FTO knockdown reduces MT-A70 expression, a subunit of m6A methyltransferase, indicating that the increase in m6A is due to FTO depletion, not increased methyltransferase activity. FTO partially colocalizes with nuclear speckles, supporting the idea that m6A in nuclear RNA is a substrate of FTO. The enzymatic alteration of m6A may be linked to processing of recently transcribed mRNA. FTO's function in demethylating m6A in mRNA suggests a novel, reversible regulatory mechanism in mammalian cells. The discovery of FTO-mediated oxidative demethylation of m6A in nuclear RNA may lead to further investigations on biological regulation based on reversible chemical modification of RNA.