Humans are widely exposed to polycyclic aromatic hydrocarbons, commonly found in cigarette smoke and diesel exhaust. These can undergo site- and cell-specific metabolism to cytotoxic intermediates. Metabolism of naphthalene and Clara cell cytotoxicity have been extensively studied in male animals. To address whether male and female mice are equally susceptible to naphthalene, mice were injected with naphthalene, and lungs were examined 1, 2, 3, 6, and 24 h after treatment. By analysis of acute injury using differential permeability to fluorescent nuclear dyes and high-resolution histopathology, injury in female mice was found to be more extensive, occur earlier, and include permeable cells in proximal airways, including airway bifurcations. HPLC analysis of the products of cytochrome P-450 (CYP)-mediated metabolism in microdissected airways indicated that although both genders produced a predominance of products from CYP2F2, female mice produced more naphthalene dihydrodiol in distal airways, the primary sites of injury. We conclude that there are clear gender differences in susceptibility to naphthalene-induced injury and that differences in metabolism of naphthalene may play a role in elevated susceptibility in female mice.