The degradation of 2-chloro-4,5-O-(4'-methyl-7', 8'-diphenyl)ether (CMDPE), an analog of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), mediated by Geobacillus sp. UZO 3 cell-free extract was monitored. Ethyl acetate extracts of a complete reaction mixture incubated at 65 degrees C for 18?h were analyzed either by thin layer chromatography (TLC) fractionation coupled with spectrometric detection or by gas chromatographymass spectrometry (GC-MS). The reaction product 4-methylumbelliferone (4MU) was successfully isolated by TLC and visualized by a transilluminator at 450?nm. The 4MU, 4-chlorophenol, and reaction intermediate 6-chlorophenoxy-4-methylumbelliferone were all successfully detected by GC-MS. The presence of these compounds suggest that Geobacillus sp. UZO 3 cell-free extract also catalyzes the reductive cleavage of the diaryl ether bonds of CMDPE in a similar mechanism previously reported in 2,7-DCDD. In the present study, the authors describe a simple and highly sensitive fluorescent assay for a new dioxin degrading enzyme(s). Environ. Toxicol. Chem. 2012; 31: 10721075. (c) 2012 SETAC