Glyceryl-ether monooxygenase (1-alkyl- sn-glycerol,tetrahydropteridine:oxygen oxidoreductase, EC 1.14.16.5) catalyzes the oxidative cleavage of 1- O-alkyl glycerol or glycol derivatives to a long-chain aldehyde and the glycerol or glycol derivative. The specificity for tetrahydropterins of a similar, perhaps identical, enzyme that cleaves O-hexadecyl ethylene glycol in rat liver microsomes was examined with the use of an assay based on [1- 3H]ethylene glycol formation from 2-hexadecyloxy[1- 3H]ethan-1-ol. Several tetrahydropterin derivatives are effective electron donors for this reaction, and 2,4,5-triamino-6-hydroxypyrimidine is somewhat effective, but NADH, NADPH, ascorbate, reduced dichlorophenolindophenol and glutathione are inactive. Tetrahydropterin derivatives differ from each other in apparent K m and apparent V max. The order of increasing apparent K m values is tetrahydropterin ≈ 6-methyltetrahydropterine ≈ tetrahydrobiopterin < 6.7-dimethyltetrahydropterin < tetrahydrofolate. The order of increasing apparent V max values is tetrahydrofolate ≈ tetrahydropterin < 6-methyltetrahydropterin ≈ tetrahydrobiopterin ≈ 6,7-dimethyltetrahydropterin. Results obtained with the use of a spectrophotometric assay, in which tetrahydropterin oxidation is coupled to NADH oxidation by dihydropteridine reductase (NAD(P)H:6,7-dihydropteridine oxidoreductase, EC 1.6.99.7), indicated that the ration of 6,7-dimethyltetrahydropterin or 6-methyltetrahydropterin oxidized to ether lipid degraded is about 1.1 to 1.3. Unlike cytochrome P-450-dependent hydroxylases, this alkyl glycol-ether monooxogenase is not inhibited by carbon monoxide. 1- O-hexadecyl- rac-glycerol (chimyl alcohol) competitively inhibits the oxidation of the glycol ether indicating that the same enzyme probably catalyzes the oxidation of both O-alkyl glycol and 1- O-alkyl glycerol.