AbstractArchaeal enzymes have great potential for industrial use; however, expressing them in their natural hosts has proven challenging. Growth conditions for many archaea are beyond typical fermentation capabilities, and to compound the problem, archaea generally achieve much lower biomass yields than Escherichia coli or Saccharomyces cerevisiae. To determine whether a eukaryotic host, S. cerevisiae, would be a suitable alternative for archaeal protein production, we examined the expression of the tetrameric β‐glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. We engineered the β‐glucosidase to facilitate secretion into the culture medium and have demonstrated the β‐glucosidase's secretion and activity. We determined the dependence of β‐glucosidase secretion on gene copy number and obtained a transformant capable of secreting ∼10 mg/L in batch culture. All transformants retained large intracellular fractions of β‐glucosidase, indicative of an intracellular bottleneck. Cell fractionation by sucrose density centrifugation and immunofluorescence identified the endoplasmic reticulum as the secretion bottleneck. Preliminary evidence indicates that the cause of this bottleneck is misfolding of the monomeric β‐glucosidase, rather than tetrameric association. Expression at moderately elevated temperatures (between 30 and 40°C) improved β‐glucosidase yields, suggesting that higher temperature expression may improve folding and secretion yields. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 713–723, 2002.