Folding of the airway mucosal membrane provides a mechanical load that impedes airway smooth muscle contraction. Mechanical testing of rabbit tracheal mucosal membrane showed that the membrane is stiffer in the longitudinal than in the circumferential direction of the airway. To explain this difference in the mechanical properties, we studied the morphological structure of the rabbit tracheal mucosal membrane in both longitudinal and circumferential directions. The collagen fibers were found to form a random meshwork, which would not account for differences in stiffness in the longitudinal and circumferential directions. The volume fraction of the elastic fibers was measured using a point-counting technique. The orientation of the elastic fibers in the tissue samples was measured using a new method based on simple geometry and probability. The results showed that the volume fraction of the elastic fibers in the rabbit tracheal mucosal membrane was approximately 5% and that the elastic fibers were mainly oriented in the longitudinal direction. Age had no statistically significant effect on either the volume fraction or the orientation of the elastic fibers. Linear correlations were found between the steady-state stiffness and the quantity of the elastic fibers oriented in the direction of testing.