In this paper, we report for the first time the functional expression of lipase B from the yeast Candida antarctica (CalB) in the Escherichia coli cytoplasm. The enzyme possessing three disulfide bonds was functionally expressed in the strain Origami B. Expression under the control of a lac promoter yielded 2 U mg(-1), whereas expression of a thioredoxin-CalB fusion protein yielded 17 U mg(-1). The native enzyme was most efficiently expressed under control of the cspA promoter (11 U mg(-1)). Coexpression of different chaperones led to a strong increase in active protein formation (up to 61 U mg(-1)). A codon-optimized synthetic variant of calb did not show significant effects on functional protein yield. Functional CalB expression was not only achieved in shake flasks but also in microtiter plate scale. Therefore, this CalB expression system is suitable for high-throughput applications, including the screening of large gene libraries as those derived from directed evolution experiments.