Previous studies have suggested that the rate of inhaled O3 absorption from the intrapulmonary gas phase is principally mediated by reaction-dependent mechanisms rather than by physical solubility, tissue diffusion, or blood flow (Postlethwait et al., 1994, Toxicol. Appl. Pharmacol. 125, 77-89). The initial site of interaction between O3 and the lung surface occurs at the gas/liquid interface of the epithelial lining fluid (ELF). Therefore, we investigated (a) whether reactive uptake by ELF constituents could account for pulmonary uptake and (b) whether selected constituents acted as O3-specific absorption targets. Rat ELF was harvested by bronchoalveolar lavage. By injecting the same lavage fluid a second [(BALF)2] and/or third [(BALF)3] time into fresh lungs, a more concentrated form of ELF was obtained. Controlled quasi-steady-state exposures (O3 in air; 30-min duration) of cell-free BALF and model substrates (reduced glutathione, GSH) were utilized. Results were based on temperature-specific fractional and normalized uptake rates (r). We observed the following: (1) Buffer pH substantially influenced O3 absorption by GSH but by BALF only modestly. (2) Uptake displayed significant [BALF] and [GSH] dependence. (3) Fractional uptake decreased (BALF and GSH) with increasing [O3] although absolute uptake increased. (4) Absorption demonstrated temperature dependence. Arrhenius plots [ln(r) vs 1/T] were used to compute activation energies (Ea) and Q10. (BALF)1 Ea = 3387 cal/g mol with Q10 = 1.20. GSH (1 mM) Ea = 2240 with Q10 = 1.13. (5) Increasing flow reduced fractional uptake in a nonlinear fashion. (6) Dialysis (1000-molecular-weight cutoff) reduced uptake by (BALF)1 moderately (-30%). Sulfhydryl depletion produced minimal effect (-10%), while ascorbate depletion (-37%) and combined sulfhydryl and ascorbate depletion (-39%) were the most effective. Treatments produced lesser effects on (BALF)3. We conclude that the pH, aqueous substrate, and temperature-dependence and the Ea and Q10 are consistent with reaction-dependent O3 uptake by ELF. The analogous absorption characteristic between the ELF and intact lung (temperature, [O3], contact time) suggests that the ELF represents the primary site for O3-reactive absorption. Reduced sulfhydryls do not appear to substantially interact with inhaled O3. Principal absorption targets may include ascorbate, phospholipids, and other moderate to large molecular weight constituents.