Several agents which antagonize or interact with Ca2+ in the excitation–contraction coupling of smooth muscle have been tested for their effects on Ca2+ accumulation by subcellular fractions of the rat uterus. Na+, but not K+, reduced the amount of Ca2+ that could be accumulated by the plasma membrane fraction (PM) and the endoplasmic reticular fraction (ER). Sr2+ considerably reduced Ca2+ accumulation by PM, ER, and the mitochondrial fraction; Ba2+ had a similar effect but was not as potent as Sr2+ La3+ at concentrations up to 10 mM was unable to block Ca2+ accumulation by PM and ER completely. The pharmacological agents D600, verapamil, and chlorpromazine all inhibited Ca2+ accumulation at dose levels much higher than those required to inhibit contractility, and were ineffective at lower doses. The action of Ca2+ antagonists is seen to be complex, involving interactions at different sites and on different processes. For a fuller understanding of their mechanisms of action further studies upon passive binding and on the release of Ca2+ from subcellular sites are required.