A two-step method, i.e., the transfer acyl analysis and then the chiral HPLC analysis, was employed in the screening of the cosmid library of Microbacterium hydrocarbonoxydans genome. Two enantiocomplementary gamma-lactamase clones were found. A 40-kb cosmid showed (-)-gamma-lactamase activity, and the activity was from Mhg which was reported previously according to the results of PCR identifying experiment. The 37-kb (+)-gamma-lactamase cosmid was further constructed into a pUC18 plasmid library and screened by the same two-step method. A plasmid clone harboring a 1.6-kb fragment showed (+)-gamma-lactamase activity. A 555-bp ORF in the 1.6-kb fragment showed high (+)-gamma-lactamase activity when it was expressed under the control of T-7 promoter. The coding protein showed significant homology with bacterial isochorismatase. The (+)-gamma-lactamase was characterized and compared with the (-)-gamma-lactamase Mhg. This is another report that two enantiocomplementary gamma-lactamases are present in the same strain.