We have prepared a temperature-sensitive Saccharomyces cerevisiae type 2A phosphatase (PP2A) mutant, pph21-102. At the restrictive temperature, the pph21-102 cells arrested predominantly with small or aberrant buds, and their actin cytoskeleton and chitin deposition were abnormal. The involvement of PP2A in bud growth may be due to the role of PP2A in actin distribution during the cell cycle. Moreover, after a shift to the non-permissive temperature, the pph21-102 cells were blocked in G2 and had low activity of Clb2-Cdc28 kinase. Expression of Clb2 from the S.cerevisiae ADH promoter in pph21-102 cells was able to partially bypass the G2 arrest in the first cell cycle, but was not able to stimulate passage through a second mitosis. These cells had higher total amounts of Clb2-Cdc28 kinase activity, but the Clb2-normalized specific activity was lower in the pph21-102 cells compared with wild-type cells. Unlike wild-type strains, a PP2A-deficient strain was sensitive to the loss of MIH1, which is a homolog of the Schizosaccharomyces pombe mitotic inducer cdc25+. Furthermore, the cdc28F19 mutation cured the synthetic defects of a PP2A-deficient strain containing a deletion of MIH1. These results suggest that PP2A is required during G2 for the activation of Clb-Cdc28 kinase complexes for progression into mitosis.