The pulmonary epithelial lining fluid (ELF) contains substrates, e.g., ascorbic acid (AH 2 ), uric acid (UA), glutathione (GSH), proteins, and unsaturated lipids, which undergo facile reaction with inhaled ozone (O 3 ). Reactions near the ELF gas/liquid interface likely provide the driving force for O 3 absorption (“reactive absorption”) and constrain O 3 diffusion to the underlying epithelium. To investigate the potential mechanisms wherein O 3 /ELF interactions may induce cellular damage, we utilized a red cell membrane (RCM) model intermittently covered by an aqueous film to mimic the lung surface compartmentation, and evaluated exposure-mediated loss of acetylcholinesterase activity (AChE) and TBARS accumulation. In the absence of aqueous reactants, O 3 exposure induced no detectable changes in AChE or TBARS. AH 2 and GSH preferentially induced oxidative damage in a dose-dependent fashion. AH 2 -mediated RCM oxidation was not inhibited by superoxide dismutase, catalase, mannitol, or Fe chelators. O 3 reaction with UA, Trolox, or albumin produced no RCM oxidation but oxidation occurred when AH 2 was combined with UA or albumin. Rat bronchoalveolar lavage fluid (BALF) also induced RCM oxidation. However, in vivo O 3 exposure dampened the extent of BALF-mediated RCM oxidation. Although we cannot completely rule out O 3 diffusion to the RCM, product(s) derived from O 3 + AH 2 /GSH reactions (possibly O 3 − or 1 O 2 ) likely initiated RCM oxidation and may suggest that in vivo, such secondary species account for O 3 permeation through the ELF leading to cellular perturbations.