The fission yeast Schizosaccharomyces pombe contains two dithiol glutaredoxins (Grx1 and Grx2) and genes for three putative monothiol glutaredoxins (grx3, 4, and 5). We investigated the expression, sub-cellular localization, and functions of the three monothiol glutaredoxins. Fluorescence microscopy revealed that Grx3 is targeted to nuclear rim and endoplasmic reticulum, Grx4 primarily to the nucleus, and Grx5 to mitochondria. Null mutation of grx3 did not significantly affect growth and resistance against various oxidants, whereas grx5 mutation caused slow growth and sensitivity toward oxidants such as hydrogen peroxide, paraquat, and diamide. The grx2grx5 double mutation, deficient in all mitochondrial glutaredoxins, caused further retardation in growth and severe sensitivity toward all the oxidants tested. The grx4 mutation was not viable, suggesting a critical role of Grx4 for the physiology of S. pombe. Overproduction of Grx3 and Grx5, but not the truncated form of Grx5 without mitochondrial target sequence, severely retarded growth as Grx2 did, supporting the idea that Grx2, 3, and 5 are targeted to organellar compartments. Our results propose a distinct role for each glutaredoxin to maintain thiol redox balance, and hence the growth and stress resistance, of the fission yeast.