Proline protects yeast cells from damage caused by various stresses. A yeast Saccharomyces cerevisiae mutant with high levels of intracellular proline grown in a minimal medium accumulated proline in its vacuole, but when grown in a nutrient medium, accumulated proline mainly in the cytosol. To understand the role of the proline pool in the vacuole, we examined the stress-protective effect of proline in proline-accumulating yeast cells deficient in vacuolar functions. The disruption of PEP3 encoding a vacuolar membrane protein required for vacuolar biogenesis caused hypersensitivity to heat shock and ethanol stresses, probably due to disappearance of normal vacuoles. The vph1-disrupted cells lacking vacuolar-ATPase activity showed resistance to heat shock without any change in proline localization, but showed severe growth defects in an ethanol-containing medium. These results indicate that vacuolar functions are involved in the stress-protective effect of proline in S. cerevisiae. Also, it appears that excess proline is transported to the vacuole in an ATP-independent manner.