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Isoflurane is metabolized by both rat and human hepatic microsomal cytochrome P-450 in vitro to fluoride ion and organofluorine metabolites. The forms of rat liver microsomal cytochrome P-450 induced by phenobarbital and pregenolone-16α-carbonitrile appear to be involved in'the metabolism of isoflurane, while the forms induced by β naphthoflavone do not. Different pathways are favored for the metabolism of isoflurane by rat and by human liver icrosomes: trifluoroacetaldehyde appears to be produced from isoflurane by rat liver microsomal cytochrome P-450, while trifluoracetate or other nonvolatile fluorinated metabolites were not. The trifluoroacetaldehyde so produced binds tightly to microsomal constituents. Human liver microsomes converted isoflurane extensively to nonvolatile fluorinated products, one of which appears to be trifluoroacetate. The proposed pathways for the metabolism of isoflurane are considered in view of the above results.