An ether bond-cleaving enzyme was purified as diglycolic acid (DGA) dehydrogenase associated with 3-(4,5-dimethyl-2-thioazolyl)-2,5-diphenyl-2H tetrazolium bromide and phenazine methosulfate. DGA dehydrogenase was inductively formed in a polyethylene glycol (PEG)-utilizing symbiotic mixed culture, E-1, composed of Sphingomonas terrae and Rhizobium sp. which were grown on PEG 6,000. DGA dehydrogenase was solubilized with 0.5% n-dodecyl-beta-D-maltoside from membrane fractions of the mixed culture E-1 and purified almost to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis, by DEAE-Toyopearl column chromatography and Sepharose 6B gel filtration. The molecular weight of the enzyme was determined to be about 40,000-41,000 Da by SDS-polyacrylamide gel electrophoresis and 480,000 by gel filtration on Sepharose 6B in the presence of 0.5% n-dodecyl-beta-D-maltoside, The absorption spectrum of the purified enzyme showed two peaks at 345 nm and 430-450 nm in addition to a major peak at 280 nm. The optimal pH and temperature were 8.9 and 40 degrees C, respectively. The enzyme was stable in the pH range of 7.8 to 9.0 and at below 30 degrees C. The enzyme was activated by neither metal ions nor ammonium ion, and strongly inhibited by 1,4-benzoquinone. The enzyme catalyzed the degradation of DGA, PEG dicarboxylic acids, glycolic acid and glyoxylic acid, but not primary alcohols, ethylene glycol (EG), EG oligomers, PEG 6,000 and aldehydes.