Fusarium moniliforme CGMCC 0536 showing excellent stereoselective D-pantonohydrolase activity was immobilized by cross-linking with glutaraldehyde. The stereospecificity of the reaction system were not affected after cross-linking. The feasibility of continuous asymmetric hydrolysis of DL-pantolactone coupled to the separation of biocatalyst on-line in a membrane bioreactor was investigated. The influence of reaction parameters like membrane cut-off and cross-flow rate was tested. Continuous D-pantoic acid production was efficient for about 48 h, followed by membrane fouling. (C) 2004 Elsevier Ltd. All rights reserved.