Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1a dagger, yro2a dagger, and mrh1a dagger yro2a dagger) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.