Abstract A temperature-sensitive mutant of Saccharomyces cerevisiae has been isolated which accumulates a large pool of trehalose-6-phosphate when shifted to temperatures above 34°C nonpermissive for growth. This indicates that its defect is in the second enzyme of trehalose biosynthesis, the hydrolase that converts trehalose-6-phosphate to trehalose. Trehalose is made continouosly when yeast is growing on high glucose or when it is starved for a nitrogen source, and accumulates as cells enter the stationary phase. Revertants of the mutant able to grow at 37°C arise spontaneously and no longer accumulate trehalose-6-phosphate at this temperature. Also the kinetics of trehalose-6-phosphate accumulation in the mutant following a 25–37°C shift resemble the kinetics of inhibition of RNA and protein synthesis. It is probable therefore that accumulation of high levels of this metabolic intermediate is inhibitory to growth.