The interaction of calcyclin with tropomyosin and tropomyosin. actin was studied with fluorescence titrations and photo-reactive crosslinking experiments. Titrations of pyreneiodoacetamide-labeled tropomyosin alone or with actin showed binding of calcyclin to tropomyosin with μM dissociation constants when Ca 2 + was present. UV irradiation of mixtures of calcyclin and gizzard ββ-tropomyosin labeled with benzophenone-iodoacetamide at Cys36, with or without actin, produced crosslinks between tropomyosin chains and calcyclin monomers only in the presence of Ca 2 + . These data provide direct evidence for a Ca 2 + -dependent tropomyosin-calcyclin interaction at or near Cys36 of tropomyosin and indicate that calcyclin binding to tropomyosin.actin does not cause tropomyosin dissociation.