Striated muscle is the primary source of biomechanical force in organisms from worms to man, and is organized as a composite material from nanometer to meter scales. At nanometer scale, an array of cytoskeletal proteins are required to regulate the size, assembly and function of the sarcomere, as well as transmit force and provide elasticity for restoring the structure. One such protein is the giant protein titin (Mr 3-4 x 10(6)), which spans half of the muscle sarcomere length. The passive elasticity of muscle at a physiological range of stretch arises primarily from the extension of titin. We have measured both the extension force and the dynamic stiffness of native titin with the atomic force microscope. We found that the stiffness of a single molecule of titin varied between 1 and 4 pN/nm over an extension of >1 mum. Regions showing globular domains unfolding in the sawtooth pattern of force versus extension curve exhibit periodic spikes in the stiffness. The results of this study show that simultaneous stiffness and force measurements of single molecules can provide new information on the elastic behavior and structural transitions of elastic proteins and their physiological roles in muscle elasticity.