Native thin filaments were extracted from rabbit uterus by the procedure of Marston and Smith. The protein content was actin, tropomyosin, and caldesmon in molar ratios of 1:0.2:0.03. Some filamin, myosin, and calcium-binding protein were also present. The thin filaments activated skeletal or smooth muscle myosin magnesium adenosine triphosphatase at least 30-fold. Activation was regulated by Ca2+; maximum observed Ca2+ sensitivity was greater than 10 times. The thin filaments were dismantled into component proteins by the method of Smith and Marston. Actin and actin-tropomyosin-activated myosin magnesium adenosine triphosphatase, but the activation was not Ca2+-regulated. Added caldesmon inhibited adenosine triphosphatase activation by as much as 80%, with 50% inhibition at 1 caldesmon per 50 actin. Caldesmon inhibition was not Ca2+ dependent, but inhibition could be reversed by further addition of Ca2+ and calmodulin. It is concluded that the thin filaments of uterine smooth muscle are Ca2+ regulated and that this regulatory system could be involved in control of uterine smooth muscle contractility. A mechanism for thin filament regulation, mediated by caldesmon, is proposed.