Difluoromethoxyldifluoracetic acid (CHF2OCF2CO2H) has been identified as a metabolite of enflurane (CHF2OCF2CHClF) in rat liver microsomes in vitro and in human urine by gas chromatography mass spectrometry. A radiochemical assay showed that this was the only organic metabolite of enflurane in the urine of these patients. The formation of the metabolite in rat liver microsomes requires NADPH and O2, and is inhibited by SKF 525-A or CO:O2 (8:2). When the C-H bonds of the CHClF groups of enflurane or the CHCl group of isoflurane (CHF2OCHClCF3) are replaced with C-Cl bonds virtually no fluoride ion (F-) is produced from either derivative in liver microsomes. These results indicate that cytochrome P-450 catalyzes the oxidative dehalogenation of CHF2OCF2CHClF at its CHClF group to form CHF2OCF2CO2H, Cl- and F-. In contrast, the CHF2 group does not appear to be appreciably susceptible to metabolic oxidative dehalogenation. Therefore, the CHF2 group can be incorporated into the design of new inhalation anesthetics so that they will not be appreciably metabolized to the potential kidney toxin, F-.