The esterase III encoded by estC gene of Pseudomonas fluorescens was overexpressed in Escherichia coli BL21(DE3) by using the T7 expression system. The open reading frame of estC gene amplified by polymerase chain reaction was subcloned into T7 promoter-containing plasmid, pRSET. The resultant plasmid, pREIII was used for transformation of E. coli cells. The expression level of esteraseIII in E. coli harboring pREIII was 20 times higher than that harboring pUE892 which contains estC under lac promoter. The protein was successfully expressed without forming any inclusion body in side the E. coli cells. The amount of the enzyme was estimated to be 7-14\% of total cellular protein as judged by densitometer after SDS-gel electrophoresis. Following sequential DEAE-Sepharose ion exchange chromatography and Superose 12 gel filtration chromatography, the protein was purified to homogeneity. After purification, activity yield was 34\% and thr specific activity was 107000.