Abstract: Reactive oxygen species (ROS) are a part of cellular homeostasis. At the same time, excessive ROS can lead to the accumulation of oxidized macromolecules such as proteins, lipids, and nucleic acids, which can be detrimental to cells. A class of proteins that can reverse some protein oxidation events are the methionine sulfoxide reductases (Msrs). The Msrs can repair oxidized methionine adducts induced by ROS, and they are considered an important means to limit oxidative damage to proteins. Methionine sulfoxide (MetO) is generated upon oxidation of the methionine side chain by ROS. MetO exists as two epimers with a chiral center at the oxidized sulfur atom. Two classes of Msrs mediate the stereospecific reduction of S- or R- MetO epimers: MsrA and MsrB. In the yeast Saccharomyces cerevisiae, these enzymes are referred to as Mxr1 and Mxr2. Mxr1 localizes to the cytoplasm and nucleus and reduces S-MetO. Mxr2 is dual-localized to the cytoplasm and mitochondria and reduces R-MetO. Why some cellular locations contain both an MsrA/Mxr1 and MsrB/Mxr2, and other locations contain a single Msr is unclear. Very few substrates of the Msrs have been identified, leaving open the question if some proteins are more susceptible to forming S- or R-MetO epimers, and if these substrates are found in specific cellular compartments. In this work, we focus on uncovering how expression of the two isoforms of Mxr2 is regulated in yeast cells, as a first step towards uncovering why the yeast cell expresses a single Msr in the mitochondria yet contains two Msrs in the cytoplasm. Prior work established that the cytoplasmic (Mxr2cyto) and mitochondrial (Mxr2mito) isoforms are generated through the alternative use of two in-frame start codons that flank a mitochondrial targeting sequence (MTS). Prior work also showed that most Mxr2 localizes to the cytoplasm. Here we reveal that the two isoforms are produced from alternative transcripts. We uncover a regulatory region upstream of the MRX2 coding region that when removed promotes the production of a longer MXR2 transcript and robust expression of Mxr2mito. These data suggest that the cell normally dampens the production of the mitochondrial isoform, and that conditions may exist wherein Mxr2mito expression is upregulated to reverse R-MetO modifications in the mitochondria. We observed that both Mxr2mito protein levels, and a corresponding long transcript, are enriched in actively respiring cells grown in non-fermentable carbon sources (ethanol and lactate). Respiring cells are associated with elevated levels of mitochondrial ROS, and these data suggest mitochondrial proteins may be more susceptible to methionine oxidation during these conditions. Overall, these data set the stage for further characterization of the conditions and mechanisms for the upregulation of Mxr2mito expression and Msr substrate identification.