Desiccation tolerance, the ability to survive nearly total dehydration, is a rare strategy for survival and reproduction observed in all taxa. However, the mechanism and regulation of this phenomenon are poorly understood. Correlations between desiccation tolerance and potential effectors have been reported in many species, but their physiological significance has not been established in vivo. Although the budding yeast Saccharomyces cerevisiae exhibits extreme desiccation tolerance, its usefulness has been hampered by an inability to reduce tolerance more than a few fold by physiological or genetic perturbations. Here we report that fewer than one in a million yeast cells from low-density logarithmic cultures survive desiccation, while 20-40% of cells from saturated cultures survive. Using this greatly expanded metric, we show that mutants defective in trehalose biosynthesis, hydrophilins, responses to hyperosmolarity, and hypersalinity, reactive oxygen species (ROS) scavenging and DNA damage repair nevertheless retain wild-type levels of desiccation tolerance, suggesting that this trait involves a unique constellation of stress factors. A genome-wide screen for mutants that render stationary cells as sensitive as log phase cells identifies only mutations that block respiration. Respiration as a prerequisite for acquiring desiccation tolerance is corroborated by respiration inhibition and by growth on nonfermentable carbon sources. Suppressors bypassing the respiration requirement for desiccation tolerance reveal at least two pathways, one of which, involving the Mediator transcription complex, is associated with the shift from fermentative to respiratory metabolism. Further study of these regulators and their targets should provide important clues to the sensors and effectors of desiccation tolerance.