Protein phosphatase 2A (PP2A) is one of the major serine/threonine phosphatases found in eukaryotic cells. We cloned two genes, par1(+) and par2(+), encoding distinct B' subunits of PP2A in fission yeast. They share 52% identity at the amino acid sequence level. Neither gene is essential but together they are required for normal septum positioning and cytokinesis, for growth at both high and low temperature, and for growth under a number of stressful conditions. Immunofluorescence microscopy revealed that Par2p has a cell-cycle-related localization pattern, being localized at cell ends during interphase and forming a medial ring in cells that are undergoing septation and cytokinesis. Our analyses also indicate that Par1p is more abundant than Par2p in the cell. Cross-organism studies showed that both par1(+) and par2(+) could complement the rts1Delta allele in Saccharomyces cerevisiae, albeit to different extents, in spite of the fact that neither contains a serine/threonine-rich N-terminal domain like that found in the S. cerevisiae homolog Rts1p. Thus, while Schizosaccharomyces pombe is more similar to higher eukaryotes with respect to its complement of B'-encoding genes, the function of those proteins is conserved relative to that of Rts1p.