The objectives of this study were to quantitatively define alterations in the epithelial populations and expression of cytochrome P450 monooxygenases in distal airways which are associated with the tolerance resulting from repeated exposure of mice to the acute Clara cell toxicant, naphthalene. Bronchiolar epithelium of mice treated daily for 7 days with an acutely cytotoxic dose of naphthalene (200 mg/kg/day) quantitatively resembles the bronchiolar epithelium of control animals in terms of proportions of ciliated and nonciliated cells and nuclear and cytoplasmic volumes. Subsequent treatment of tolerant animals with higher doses (300 mg/kg) does not produce the same Clara cell injury observed in previously untreated mice after a single treatment with 300 mg/kg. After repeated exposures to naphthalene, cellular expression of immunoreactive cytochrome P450 monooxygenases 2B and 2F, P450 reductase and Clara cell secretory protein significantly decreased in bronchiolar epithelium. Although metabolism of naphthalene to the 1R,2S-naphthalene oxide is depressed in microsomes derived from whole-lung homogenates of tolerant animals, metabolism of naphthalene in distal airways isolated by microdissection is unchanged from the controls at saturating substrate concentrations. When substrate concentrations are less than the apparent K(m) for naphthalene (< 0.05 mM), the rate of naphthalene metabolite production is markedly lower in distal airways of tolerant mice compared with controls. Covalent binding of reactive naphthalene metabolites in lungs of tolerant mice is also unchanged from control. In conclusion, after repeated exposure to short-term cytotoxic doses of naphthalene distal bronchioles of tolerant mice 1) are lined by epithelium which resembles that of controls; 2) express lower levels of P450 proteins; 3) have reduced levels of naphthalene monooxygenase activity, but only at less than saturating concentrations and 4) have no decrease in covalent binding of reactive metabolites to proteins.