The CYP2F enzymes, abundantly expressed in the respiratory tract, are active toward many xenobiotic compounds, including naphthalene (NA). However, the precise roles of these enzymes in tissue-selective chemical toxicity have been difficult to resolve. A Cyp2f2-null mouse was generated in this study by disrupting the Cyp2f2 fourth exon. Homozygous Cyp2f2-null mice, which had no CYP2F2 expression and showed no changes in the expression of other P450 genes examined, were viable and fertile and had no in utero lethality or developmental deficits. The loss of CYP2F2 expression led to substantial decreases in the in vitro catalytic efficiency of microsomal NA epoxygenases in lung (up to ~160-fold), liver (~3-fold), and nasal olfactory mucosa (OM; up to ~16-fold), and significant decreases in rates of systemic NA (300 mg/kg i.p.) clearance. The Cyp2f2-null mice were largely resistant to NA-induced cytotoxicity, when examined at 24 h after NA dosing (at 300 mg/kg i.p.), and to NA-induced depletion of total nonprotein sulfhydryl (NPSH), examined at 2 h after dosing, in the lungs. In contrast, the loss of CYP2F2 expression did not alleviate NA-induced NPSH depletion or tissue toxicity in the OM. Mouse CYP2F2 clearly plays an essential role in the bioactivation and toxicity of NA in the lung but not in the OM. The Cyp2f2-null mouse should be valuable for studies on the role of CYP2F2 in the metabolism and toxicity of numerous other xenobiotic compounds and for future production of a CYP2F1-humanized mouse.