Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that gamma-glutamylcysteine synthetase (gamma-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a gamma-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of gamma-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium.