beta-Glucosidase from Cellvibrio gilvus was successfully overproduced in soluble form in Escherichia coli, with the coexpression of GroEL/ES. Without the GroEL/ES protein, the beta-glucosidase overexpressed in E. coli constituted a huge amount (80%) of the total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the beta-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. In addition, the synergistic effect of GroEL/ES and the low induction temperature led to 70% solubilization of the total expressed target protein and more than a 20-fold increase in activity. Similar effects of GroEL/ES were also observed on the overexpressed beta-glucosidase from Agrobacterium tumefaciens.