Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus h-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to h-glucosidase misfolding. By increasing expression temperature from 30 -C up to 40 -C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 -C. We examined the effect of temperature on h-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble h-glucosidase. Likewise, stress on the cell caused by h-glucosidase expression was found to be greatly reduced at 37 -C compared to 30 -C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the h-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.