To date, quantitative studies of the inherent characteristics of the developing airway wall have required excision of an airway segment or surgical creation of an isolated segment. We hypothesized that airway wall characteristics, at various collapsing pressures, and attendant changes in stiffness after smooth muscle stimulation could be quantitated bronchoscopically from airway pressure-area relationships. Neonatal lamb tracheal segments (n = 12) were suspended over hollow mounts, in a buffer-filled chamber, and subjected to a range (0 to -4.0 kPa) of pressures to determine wall stiffness under collapsing forces before and after stimulation of the trachealis with methacholine. Luminal images were recorded through a 3.6-mm flexible bronchoscope under the same conditions, subsequently corrected for distortion, and a cross-sectional area was quantitated. Both pressure-volume and pressure-area relationships detected significant changes in airway wall stiffness after methacholine administration (p < 0.002), and the magnitude of change was similar between methods. These data suggest that quantitative flexible bronchoscopy can be used clinically in the intact airway to assess wall stiffness.