Previous studies have shown variability in naphthalene cytotoxicity, expression of CYP2F2 gene and protein, and naphthalene metabolism in random cycling female mice (NIH:Swiss). CYP2F2 metabolizes naphthalene to cytotoxic metabolites in lungs of mice. This study was designed to address the question: do hormonal changes associated with the estrous cycle alter metabolism of naphthalene in the lung? Adult virgin female mice were manipulated into defined stages of the reproductive cycle: estrus, proestrus, and noncycling. Cycling was confirmed by cytology on vaginal swabs. At specific cycle times, extrapulmonary (tracheal and bronchial) and intrapulmonary (bronchiolar) conducting airways were microdissected from the lung parenchyma and incubated with naphthalene, and the products of naphthalene metabolism were trapped and measured using high-performance liquid chromatography. Circulating estradiol levels were measured at necropsy using an enzyme-linked immunosorbent assay. CYP2F2 gene expression was determined by airway level using real-time reverse transcription-polymerase chain reaction and did not vary by estrous cycle stage in intrapulmonary airways but did in extrapulmonary airways. Metabolism of naphthalene varied significantly by estrous cycle stage with the highest level of total metabolism occurring in proestrus (when estrogen is lowest) in intrapulmonary airways. Total activity and metabolite profiles in both extrapulmonary and intrapulmonary airways were affected by cycle stage. We conclude that the hormonal patterns associated with different stages of the estrous cycle 1) alter metabolism of naphthalene in the lungs of mice and 2) alter naphthalene metabolism differentially in extrapulmonary versus intrapulmonary airways.