The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In 32P-labeled muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [g-32P, b-32P] ATP; The specific radioactivity of actin-bound ADP was equal to that of the b-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the g-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K+, as compared to resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.