A metagenomic library of surface-water microbes from the Yangtze River in China was constructed, and a novel esterase, designated as EstY, was isolated and characterized. EstY had 423 amino acids with an estimated molecular mass of 44 kDa and pI of 7.28. It hydrolyzed various pnitrophenyl esters (acetate, butyrate, caprate, caprylate, laurate, myristate, and palmitate) and its best substrate was p-nitrophenyl caprate (C8). The optimum pH for EstY activity was 9.0 and the optimum temperature was 50 degrees . Metal ions, such as Mn2+, Co2+, Hg2+, Zn2+, and Fe3+, strongly inhibited the activity of EstY, whereas Mg2+ was required for maximal activity. Activity remained in the presence of 10% alcohol, acetone, isopropanol, and dimethyl sulfoxide, respectively. An analysis of the amino acid sequence deduced from estY revealed that it had 7 closely related lipolytic enzymes. Moreover, a sequence analysis showed that EstY, like its 7 relatives, did not belong to any known lipolytic enzyme family.