Hyperventilation-induced bronchoconstriction (HIB) is a component of exercise-induced asthma (EIA) believed to result from the penetration of unconditioned air into the lung periphery. We used a canine model of EIA to examine the effect of hyperventilation on airway surface fluid (ASF) volume and osmolality, and to determine if the observed kinetics support the hypothesis that hyperventilation-induced changes in ASF osmolality initiate bronchoconstriction. Exposure of sublobar airways to dry air at baseline insufflation resulted in stable measurements of ASF volume, ASF osmolality, and peripheral airway resistance (Rp). Baseline insufflation of warm humidified air increased ASF volume, but did not alter ASF osmolality. Hyperventilation challenge with warm humidified air (WAC) increased Rp and ASF volume, but decreased ASF osmolality. Dry air challenge (DAC) increased Rp, ASF volume, and ASF osmolality. ASF osmolality during DAC was markedly higher when compared with posthyperventilation values. Post-DAC changes in (Delta) ASF volume and osmolality were poorly correlated with the development of HIB. In contrast to Delta ASF after DAC, Delta ASF osmolality during DAC was strongly correlated with HIB, and tended to be inversely related to Delta ASF volume. These observations are consistent with the hypothesis that changes in airway osmolality during hyperventilation initiate peripheral airway constriction.