The methyl donor S‐adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Methionine adenosyltransferase II is ubiquitously expressed, whereas isoenzymes I (homotetramer) and III (homodimer) are considered the hepatic enzymes. In this work, we identified methionine adenosyltransferase I/III in most rat tissues, both in the cytoplasm and the nucleus. Nuclear localization was the preferred distribution observed in extrahepatic tissues, where the protein colocalizes with nuclear matrix markers. A battery of mutants used in several cell lines to decipher the determinants involved in methionine adenosyltransferase subcellular localization demonstrated, by confocal microscopy and subcellular fractionation, the presence of two partially overlapping areas at the C‐terminal end of the protein involved both in cytoplasmic retention and nuclear localization. Immunoprecipitation of coexpressed FLAG and EGFP fusions and gel‐filtration chromatography allowed detection of tetramers and monomers in nuclear fractions that also exhibited S‐adenosylmethionine synthesis. Neither nuclear localization nor matrix binding required activity, as demonstrated with the inactive F251D mutant. Nuclear accumulation of the active enzyme only correlated with histone H3K27 trimethylation among the epigenetic modifications evaluated, therefore pointing to the necessity of methionine adenosyltransferase I/III to guarantee the supply of S adenosylmethionine for specific methylations. However, nuclear monomers may exhibit additional roles.—Reytor, E., Pérez‐Miguelsanz, J., Alvarez, L., Pérez‐Sala, D., Pajares, M. A. Conformational signals in the C‐terminal domain of methionine adenosyltransferase I/III determine its nucleocytoplasmic distribution. FASEB J. 23, 3347–3360 (2009). www.fasebj.org