The purpose of this study was to quantitate the structural changes in the airways of sensitized rats after repeated challenge with aerosolized antigen and to examine the relationship between these changes and alterations in responsiveness to methacholine (MCh). We studied 28 Brown Norway rats that were actively sensitized to ovalbumin (OA). Responsiveness to aerosolized MCh was quantitated as the concentration of MCh required to double pulmonary resistance (EC200 RL). The EC200 RL was determined before and 1 and 5 days after three inhalational challenges with OA (n = 17) or saline (n = 11) at 5-day intervals (on Days 14, 19, and 24 after sensitization). Responsiveness to MCh increased after OA; EC200 RL fell from 1.71 to 0.71 mg/ml at 1 day (p less than 0.01) and 0.87 mg/ml at 5 days (p less than 0.02) after OA but did not change after saline challenge. Formalin-fixed lungs from a sample of OA-challenged (n = 12) and saline-challenged (n = 6) animals were paraffin embedded, and 5-microns sections were stained with hematoxylin-phloxin-saffron. Cross-sectional areas of the airway wall and smooth muscle (ASM) were determined for all intrapulmonary membranous airways. There was an approximately twofold increase in the quantity of airway smooth muscle in airways of OA-challenged animals compared with saline-challenged control animals. Airway wall area did not change significantly. There was a correlation (r = 0.618, p less than 0.05) between the quantity of ASM in large airways (basement membrane length 2.00 to 2.99 mm) and change in responsiveness to MCh.