A key molecule of sensing machineries essential for survival upon hypo-osmotic shock is the mechanosensitive channel. The bacterial mechanosensitive channel MscS functions directly for this purpose by releasing cytoplasmic solutes out of the cell, whereas plant MscS homologues are found to function in chloroplast organization. Here we show that the fission yeast MscS homologues, designated Msy1 and Msy2, participate in the hypo-osmotic shock response by a mechanism different from that operated by the bacterial MscS. Upon hypo-osmotic shock, msy2(-) and msy1(-) msy2(-) cells display greater cell swelling than wild-type cells and undergo cell death. Cell swelling precedes an intracellular Ca(2+) increase, which was greater in msy1(-) and msy1(-) msy2(-) cells than in wild-type cells. Fluorescent microscopy showed that Msy1 and Msy2 localize mainly to the endoplasmic reticulum. These observations suggest that organellar Msy1 and Msy2 regulate intracellular Ca(2+) and cell volume for survival upon hypo-osmotic shock.