We used a rat pup model to delineate whether mechanical ventilation of ≤4 h duration in the absence of supplemental oxygen contributes to the development of airway hyperreactivity. Eight-day-old rat pups were assigned to unventilated normoxic controls, ventilated under normoxic conditions, ventilated under hyperoxic conditions (100% O2), or unventilated hyperoxic groups (>95% O2). After each intervention, they were returned to their mothers. On d 10 of life, all animals were anesthetized, paralyzed, and ventilated to measure pulmonary function. Total lung resistance (RL) and dynamic lung compliance (Cdyn) were measured in response to increasing intravenous doses of methacholine (0.03–1 μg/g) by head-out body plethysmography. Injection of methacholine caused a dose-dependent increase in RL and decrease in Cdyn. The response of both RL and Cdyn to methacholine was significantly potentiated by prior exposure to mechanical ventilation when compared with unventilated normoxic controls. The addition of hyperoxia to mechanical ventilation did not further potentiate responses to methacholine. Mechanical ventilation did not alter lung myosin or the number of inflammatory cells in airways of room air ventilated versus unventilated control animals. We conclude that a brief period of mechanical ventilation in rat pups increases airway reactivity 48 h after such exposure in the presence as well as absence of hyperoxic exposure. This represents a potentially important model to investigate the mechanisms involved in airway hyperreactivity induced by neonatal lung injury.