In a search for additional Ca2+ regulatory components in vascular smooth muscle, a novel troponin T-like protein was purified from bovine aorta smooth muscle. The isolated protein was separated into several isoforms on isoelectric focusing. The major isoelectric variants were focused in the pH region of 8.4 to 9.1. The protein had slightly different molecular masses in the Mr range of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molar ratio relative to tropomyosin in the muscle extract was estimated to be 0.9:1.0. The novel protein bound to the immobilized calmodulin and exhibited a number of common physicochemical properties with gizzard (Mr = 34,000) calmodulin-binding and F-actin-binding protein. The aorta and gizzard proteins were immunologically cross-reactive. Both proteins shared a common antigenic determinant with COOH-terminal segments of rabbit skeletal and bovine cardiac troponin T and bound to the immobilized smooth muscle tropomyosin. Both proteins interacted with rabbit skeletal troponin C in the presence and absence of Ca2+, but they did not interact with troponin I. These results suggest that the novel protein, which is designated calponin, may be a specialized component of smooth muscle thin filament involved in the regulation of contractile apparatus.