Airway responsiveness to methacholine and other bronchoconstrictors is highly variable within and among species. The aim of the experiments in this report was to evaluate the importance of the quantity of airway smooth muscle as a determinant of intra- and inter-species variability in airway responsiveness. To do this we established concentration-response curves to methacholine in a sample of normal guinea pigs as well as in rat, rabbit, and dog. After challenge we excised the lungs for the quantitation of smooth muscle by morphometry. Animals were anesthetized with pentobarbital and mechanically ventilated using a Harvard ventilator. Aerosols of methacholine were administered in progressively doubling concentrations from 0.0625 to 256 mg/mL for a period of 30 s for each concentration. The maximal response, determined from pulmonary resistance (RL), and the concentration of methacholine required to effect 50% of the maximal RL were determined. After provocation testing the lungs were removed and fixed with 10% Formalin. Midsagittal sections and parahilar sections were stained with hematoxylin-phloxine-saffron for microscopic examination of smooth muscle. The images of all airways in the sections were traced using a camera lucida side-arm attachment and digitized using commercial software. The area of the airway wall occupied by smooth muscle was determined and standardized for airway size by dividing it by the square of the epithelial basement membrane length. The variability in airway smooth muscle in the intraparenchymal airways was significantly greater between than within individual guinea pigs (n = 13). This was not true of extraparenchymal airways.(ABSTRACT TRUNCATED AT 250 WORDS)