Isoflurane attenuates the reduction of zona occludens 1 (ZO-1) following lipopolysaccharide and cyclic stretch in vitro. Mouse lung epithelial cells were plated in six-well dishes on flexible silico-elastic membranes and grown to confluence before treatment with lipopolysaccharide (LPS) and interferon gamma (IFN-γ). After LPS/IFN-γ treatment, cells were exposed to isoflurane (iso) (or control gas) 1 day before cyclic stretch (10% stretch, 2 h). Complementary DNA was synthesized, and message levels were analyzed by quantitative polymerase chain reaction after normalizing for the housekeeping gene 18S. In separate experiments, protein was isolated and tight junction protein levels were probed by immunoblotting. (A) The combination of LPS/IFN-γ and cyclic stretch (denoted LPS/stretch, n = 13 wells) decreased ZO-1 message levels compared with control cells (no LPS/IFN-γ and no stretch, n = 8 wells). The decrease in ZO-1 message was significantly attenuated by isoflurane treatment (n = 15 wells, *P < 0.05 vs. no isoflurane by Mann–Whitney test). (B) Occludin and claudin 4 message levels did not change significantly following the combination of LPS/IFN-γ + stretch compared with control cells (no LPS/IFN-γ, no stretch) (C) ZO-1 protein levels, but not occludin protein levels, decreased following LPS/IFN-γ + stretch. (D) Image quantitation revealed that ZO-1 expression (C) decreased significantly with LPS/IFN-γ + stretch, but ZO-1 expression did not differ significantly from control (no LPS/IFN-γ, no stretch) levels when cells were treated with isoflurane (**P < 0.05 vs. control/no iso by Kruskal–Wallis test with Dunn multiple comparisons test). All data n = 3 wells/group unless otherwise noted.