Presently, unrestrained plethysmography is widely used to assess bronchial responsiveness in mice. An empirical quantity known as enhanced pause is derived from the plethysmographic box pressure [P(b)(t), where t is time] and assumed to be an index of bronchoconstriction. We show that P(b)(t) is determined largely by gas conditioning when normal mice breathe spontaneously inside a closed chamber in which the air is at ambient conditions. When the air in the chamber is heated and humidified to body conditions, the changes in P(b)(t) are reduced by about two-thirds. The remaining changes are thus due to gas compression and expansion within the lung and are amplified when the animals breathe through increased resistances. We show that the time integral of P(b)(t) over inspiration is accurately predicted by a term containing airway resistance, functional residual capacity, and tidal volume. We conclude that unrestrained plethysmography can be used to accurately characterize changes in airway resistance only if functional residual capacity and tidal volume are measured independently and the chamber gas is preconditioned to body temperature and humidity.