To explore the role of titin filaments in muscle elasticity, we measured the resting tension-sarcomere length curves of six rabbit skeletal muscles that express three size classes of titin isoform. The stress-strain curves of the split fibers of these muscles displayed a similar multiphasic shape, with an exponential increase in tension at low sarcomere strain followed by a leveling of tension and a decrease in stiffness at and beyond an elastic limit (yield point) at higher sarcomere strain. Significantly, positive correlations exist between the size of the expressed titin isoform, the sarcomere length at the onset of exponential resting tension, and the yield point of each muscle. Immunoelectron microscopic studies of an epitope in the extensible segment of titin revealed a transition in the elastic behavior of the titin filaments near the yield point sarcomere length of these muscles, providing direct evidence of titin's involvement in the genesis of resting tension. Our data led to the formulation of a segmental extension model of resting tension that recognizes the interplay of three major factors in shaping the stress-strain curves: the net contour length of an extensible segment of titin filaments (between the Z line and the ends of the thick filaments), the intrinsic molecular elasticity of titin, and the strength of titin thick filament anchorage. Our data further suggest that skeletal muscle cells may control and modulate stiffness and elastic limit coordinately by selective expression of specific titin isoforms.