Methionine sulphoxide reductase A (MSRA) that reduces methionine-S-sulphoxide back to methionine constitutes a catalytic antioxidant mechanism to prevent oxidative damage at multiple sub-cellular loci. This study examined the relative importance of protection of the cytoplasm and mitochondria by MSRA using A-10 vascular smooth muscle cells, a cell type that requires a low level of reactive oxygen species (ROS) for normal function but is readily damaged by higher concentrations of ROS. Adenoviral over-expression of human MSRA variants, targeted to either mitochondria or the cytoplasm, did not change basal viability of non-stressed cells. Oxidative stress caused by treatment with the methionine-preferring oxidizing reagent chloramine-T decreased cell viability in a concentration-dependent manner. Cytoplasmic MSRA preserved cell viability more effectively than mitochondrial MSRA and co-application of S-methyl-L-cysteine, an amino acid that acts as a substrate for MSRA when oxidized, further increased the extent of protection. This suggests an important role for an MSRA catalytic antioxidant cycle for protection of the cytoplasmic compartment against oxidative damage.