To assess whether the changes in airway methacholine (Meth) responsiveness induced by an initial hypertonic challenge determine the response to a subsequent hypertonic provocation, 11 subjects with asthma had bronchial challenges with 3.6% hypertonic saline (HS) and Meth in a dose-dependent manner and in random order. Challenges consisted of (1) an HS challenge (HS1) followed 1 hour later by a second HS challenge (HS2), (2) a Meth challenge alone (Meth1), and (3) an HS challenge followed 1 hour later by a Meth challenge (Meth2). The dose of HS that produced a 35% fall in SGaw (PD35) in HS1 was 69 L (geometric mean), and the PD35 in HS2 was 107 L (p = 0.02). Refractory index (PD35 HS2/PD35 HS1) ranged from 0.7 to 5.0. After HS challenge, airway responsiveness to Meth increased, and the Meth PD35 fell from 0.26 mumol to 0.11 mumol (geometric mean, p = 0.004). There was an inverse linear correlation between the refractory index and increases in Meth sensitivity (PD35 Meth1/PD35 Meth2) (r = -0.66; p = 0.027). After an initial HS challenge, the ratio of PD35 HS to PD35 Meth increased in all subjects, indicating that all subjects had become less responsive to HS compared to Meth, irrespective of their refractory index. We suggest that an initial HS challenge induces protective mechanisms toward a subsequent HS challenge in all individuals. The degree of increase in Meth responsiveness elicited by the initial provocation is a major factor in determining the airway response to a subsequent HS challenge.