Plate assays were developed for the identification of specific hydrolytic activities of esterases from Burkholderia gladioli, cloned and expressed in Escherichia coli. Clones showing different substrate specificities were identified by fluorescence or azo-dye formation caused by the released alcohol moiety of the hydrolyzed substrates, or by colour change of pH indicators mediated by decreased pH. The use of 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS-) esters and linalyl acetate for these assays clearly allowed to discriminate substrate specificities for two different cloned esterases, EP6 and EP10. Long chain fatty acid HPTS-esters were only hydrolyzed by the EP10 clone. On the other hand, the EP6 clone showed significant activity in hydrolysis of the sterically hindered ester linalyl-acetate. Enantioselective hydrolysis of linalyl acetate could be verified with a crude EP6 preparation on a preparative scale.