Injury to pulmonary epithelial cells from chemicals that undergo P450-dependent metabolic activation and from gases such as ozone is highly focal. These studies examined the rates of glutathione resynthesis in pulmonary subcompartments (trachea, minor daughter/respiratory bronchiole and parenchyma) of mice and monkeys to determine whether differences in glutathione synthesis are partly responsible for wide regional/species variations in susceptibility of the lung to insult. Glutathione levels remained unchanged in lung subcompartments incubated for up to 4 hr in Waymouth's medium. Glutathione levels decreased less than 30% in 4-hr incubations of monkey airways in medium devoid of sulfur amino acids although in mouse airways decreases of 40 to 60% were observed. Diethyl maleate depleted glutathione in lung subcompartments in vitro by varying amounts depending on the species and subcompartment examined. Airways incubated in the presence of cysteine but not methionine or glutathione regenerated glutathione rapidly after diethyl maleate depletion. The rates of regeneration differed significantly with species and airway level. In all airways of the monkey, glutathione levels returned to the initial level within 2 to 4 hr after addition of cysteine containing medium although in mice recovery of glutathione required only 1 (minor daughter and parenchyma) or 2 hr (trachea) incubation with cysteine supplemented medium. These studies show striking species and airway level differences in the rates of glutathione resynthesis and suggest that focal injury to respiratory epithelium may, in part, be mediated by regional differences in the ability to supply glutathione for protection against electrophiles and reactive oxygen species.