Objectives: The mechanical property of immobilized joints is not well understood. The present study was designed to investigate the tissue elasticity of the anterior and posterior synovial membrane (SM) in a rat immobilized knee model using scanning acoustic microscopy (SAM). Moreover, the structural characteristics of the SM after immobilization were examined by transmission electron microscopy (TEM). Methods: Thirty rats had their knee joints immobilized with a plate and metal screws. The rats were fixed at 1, 2, 4, 8 and 16 weeks after surgery and the knee joints were sectioned sagittally for SAM. Selected specimens were processed for TEM. A new concept SAM using a single pulsed wave instead of continuous waves was applied to measure the sound speed of the anterior and posterior SM, comparing it with the corresponding light microscopic images. Results: The sound speed of the posterior SM increased significantly in the 8- and 16-week experimental group compared with that in the control group. The sound speed of the anterior SM showed no statistical difference between the experimental and the control groups at any period of immobilization. The posterior SM of the experimental group was different from that of the control group in the ultrastructural characteristics of extracellular matrices. Conclusions: Our data suggest that the increased elasticity and structural changes of the posterior SM are one of the main causes of limited extension after a long period of immobilization in flexion using SAM, which is a powerful tool for evaluating the elasticity of targeted tissues.