The phosphorylation status of cellular proteins results from an equilibrium between the activities of protein kinases and protein phosphatases (PPases). Reversible protein phosphorylation is an important aspect of signal transduction that regulate many biological processes in eukaryotic cells. The Saccharomyces cerevisiae genome encodes 40 PPases, including seven members of the protein phosphatase 2C subfamily (PTO to PTC7). In contrast to other PPases, the cellular roles of Pits have not been investigated in detail. Here, we sought to determine the cellular role of PTC6 in S. cerevisiae with disruption of PTC genes. We found that cells with Delta ptc6 disruption were tolerant to the cell wall-damaging agents Congo red (CR) and calcofluor white (CFW); however, cells with simultaneous disruption of PTC1 and PTC6 were very sensitive to these agents. Thus, simultaneous disruption of PTC1 and PTC6 gave a synergistic response to cell wall damaging agents. The level of phosphorylated Slt2 increased significantly after CR treatment in Delta ptc1 cells and more so in Delta ptc1 Delta ptc6 cells; therefore, deletion of PTC6 enhanced Slt2 phosphorylation in the Delta ptc1 disruptant. The level of transcription of KIWI upon exposure to CR increased to a greater extent in the Delta ptc1 Delta ptc6 double disruptant than the Delta ptcl single disruptant. The Delta ptcl Delta ptc6 double disruptant cells showed normal vacuole formation under standard growth conditions, but fragmented vacuoles were present in the presence of CR or CFW. Our analyses indicate that S. cerevisiae PTC6 participates in the negative regulation of Slt2 phosphorylation and vacuole morphogenesis under cell wall stress conditions. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.