The differential enhanced degradation of cis- and trans-1,3-D was observed in the previous two studies performed by Ou et al. (1995) and especially Chung et al. (1999). This study was initiated to investigate the involvement of microorganisms in the differential enhanced degradation of the chemicals. As expected, microorganisms were responsible for the enhanced degradation of the chemicals. A mixed bacterial culture capable of degrading 1,3-D was isolated from an enhanced soil sample collected from a site treated with 1,3-D. Similar to the enhanced soil, the mixed culture degraded trans-1,3-D faster than cis-1,3-D. This mixed culture could not utilize cis- and trans-1,3-D as a sole source of carbon for growth. Rather, a variety of second substrates were evaluated to stimulate the differential enhanced degradation of the two isomers. As a result, the mixed culture degraded cis- and trans-1,3-D only in the presence of a suitable second substrate. Second substrates that had the capacity to stimulate the degradation included soil leachate, tryptone, tryptophan, and alanine. Other substrates tested, including soil extract, glucose, yeast extract, and indole (ailed to stimulate the degradation of the two isomers. Therefore, it appeared that the degradation of cis- and trans-1,3-D was a cometabolic process. The mixed culture was composed of four morphologically distinctive bacterial colonies.