The pulmonary epithelial lining fluid (ELF) contains substrates, e.g., ascorbic acid (AH₂), uric acid (UA), glutathione (GSH), proteins, and unsaturated lipids, which undergo facile reaction with inhaled ozone (O₃). Reactions near the ELF gas/liquid interface likely provide the driving force for O₃ absorption (“reactive absorption”) and constrain O₃ diffusion to the underlying epithelium. To investigate the potential mechanisms wherein O₃/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, O₃ exposure induced no detectable changes in AChE or TBARS. AH₂ and GSH preferentially induced oxidative damage in a dose-dependent fashion. AH₂-mediated RCM oxidation was not inhibited by superoxide dismutase, catalase, mannitol, or Fe chelators. O₃ reaction with UA, Trolox, or albumin produced no RCM oxidation but oxidation occurred when AH₂ was combined with UA or albumin. Rat bronchoalveolar lavage fluid (BALF) also induced RCM oxidation. However, in vivo O₃ exposure dampened the extent of BALF-mediated RCM oxidation. Although we cannot completely rule out O₃ diffusion to the RCM, product(s) derived from O₃ + AH₂/GSH reactions (possibly O₃⁻ or ¹O₂) likely initiated RCM oxidation and may suggest that in vivo, such secondary species account for O₃ permeation through the ELF leading to cellular perturbations.