Fluorescent labeling, limited proteolysis, amino acid sequence determinations, affinity chromatography and specific chemical crosslinking were used to determine the smallest fragment of gizzard caldesmon that interacts with actin. The time course of cleavage with thrombin or submaxillaris arginase-C protease indicates that 90kDa and 35kDa fragments are the two major pieces of the 120kDa native protein. Amino acid sequence determination indicates that the 90kDa fragment is the N-terminal portion of the molecule. Further degradation gave rise to a 15kDa product whose N-terminal amino acid sequence was determined within the first 28 amino acids. Carbodiimide crosslinking with actin revealed that the 15kDa part of the molecule is probably not involved in the actin binding process but may participate in a twisting of the F-actin filament and be responsible of the caldesmon regulatory function during smooth muscle contraction.