Stiffness of the series elastic component (SEC) of canine tracheal smooth muscle in isotonic contraction and relaxation was measured by applying small force perturbations to the muscle and measuring the resulting length perturbations. The quick, elastic length transient was taken as the change in length of the SEC (delta L). The force perturbation was a train of 10-Hz rectangular force waves varying from 0 to 10% maximum isometric tension (Po) in magnitude (delta P = 10% Po). Stiffness of the SEC was estimated by the ratio delta P/delta L. The change in SEC stiffness with respect to the change in muscle length was further studied by obtaining the stress-strain curves of the SEC at different muscle lengths using the load-clamping method. The clamps were applied at a fixed time (10 s after stimulation). Length of the muscle 10 s after contraction was controlled by the magnitude of the isotonic afterload. It was found that the apparent SEC stiffness increased as muscle length decreased. This stiffness increase is not likely due to an increase in the number of attached cross bridges, but it is probably due to the gradual diminution of the SEC length itself during muscle shortening.