Purpose: Dry-air challenges increase the osmolarity of fluid lining the luminal surface of the proximal airway. The homeostasis of surface fluid is thought to be essential for voice production and laryngeal defense. Therefore, the authors hypothesized that viable vocal fold epithelium would generate a water flux to reduce an osmotic challenge (150 mOsm mannitol) on the lumen. Bidirectional transepithelial water fluxes were measured in vocal folds exposed to physiologically realistic luminal osmotic perturbations in vitro. Method: Thirty-six native ovine vocal folds were exposed to either luminal hyperosmotic or isosmotic perturbations. Vocal fold viability and water fluxes toward the lumen and into the mucosa were measured at prechallenge baseline and for 30 min after challenge. Results: Vocal fold electrophysiological viability was maintained for the duration of osmotic perturbation. Luminal osmotic exposure increased luminally directed transepithelial water fluxes in 60% of vocal folds. This increase was electrically silent, of short duration, and would not negate the osmotic gradient. Conclusion: Ovine vocal fold epithelia detect osmotic perturbations to the luminal surface in vitro. This ability to detect and respond to changes in surface composition may be important in homeostatic regulation of vocal fold surface fluid during osmotic perturbations in respiration and phonation.