Specific thrombin proteolysis of native 120-kDa gizzard caldesmon gave rise to a major cleavage into an N-terminal 90-kDa and a C-terminal 35-kDa fragment. Fluorescent labeling, cosedimentation, passage through an affinity column, and carbodiimide crosslinking with actin revealed that the 35-kDa purified segment of the molecule contains the actin and the calcium-calmodulin binding regions. Electron microscopic analysis of its actin complex demonstrated that the 35-kDa segment possesses the bundling properties of the intact molecule. Thus, a possible pathway for the expression of the caldesmon regulatory function during smooth muscle contraction would be a conformational change twisting the helicoïdal structure of the actin filament, which occurs when the 35-kDa caldesmon portion binds to it.