AbstractYeasts used in bread making are exposed to air‐drying stress during dried yeast production processes. To clarify the genes required for air‐drying tolerance, we performed genome‐wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air‐drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air‐drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H+‐ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air‐drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air‐drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H+‐ATPase, suggesting that vacuolar H+‐ATPase helps maintain intracellular pH homeostasis, which is affected by air‐drying stress. To determine the effects of air‐drying stress on mitochondria, we analysed the mitochondrial membrane potential under air‐drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air‐drying stress, suggesting that a mitochondrial function is required for tolerance to air‐drying stress. We also analysed the correlation between oxidative‐stress sensitivity and air‐drying‐stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air‐drying stress, although ROS‐scavenging systems are not necessary for air‐drying stress tolerance. Copyright © 2008 John Wiley & Sons, Ltd.