Sod2 is the Na(+)/H(+) exchanger of the fission yeast Schizosaccharomyces pombe that is principally responsible for salt tolerance. We examined the role of nine polar, membrane associated amino acids in the ability of the protein to confer salt tolerance in S. pombe. Wild type sod2 protein with a C-terminal GFP tag effectively rescued salt tolerance in S. pombe with deleted endogenous sod2. Sod2 protein with the mutations P163A, P183A, D298N, D389N, E390Q, E392Q and E397Q also conveyed salt tolerance as effectively as the wild type sod2 protein. In contrast, the mutation P146A resulted in a protein that did not convey salt tolerance nearly as effectively as the wild type and did not extrude Na(+) as well as the wild type. Mutation of Pro(146) to Ser, Asp or Lys had an intermediate effect. Mutation of Thr(142) to Ser resulted in a slightly defective protein. Western blot analysis showed that all mutant proteins were expressed at similar levels as wild type sod2 protein. Examination of the localization of the proteins showed that wild type and most sod2 mutants were present in the plasma membrane while the P146A mutant had an intracellular localization. Limited tryptic digestion suggested that the P146A sod2 protein had a change in conformation in comparison to the wild type protein. The results suggest that Pro(146) is an amino acid critical to sod2 structure, function and localization.