The calcium content and distribution of the rat uterus were determined employing flame photometry and Ca45 determinations. The total uterine calcium concentration was found to be 2.25 millimoles (mmoles) per kilogram wet weight, 0.45 of which was inexchangeable. The exchangeable Ca could be divided into 0.8 mmole/kg wet weight extracellular and 1.0 mmole/kg wet weight intracellular. The concentration of ionic Ca in rat serum was obtained by equilibrium dialysis as 1.5 mM or 53 % of the total serum Ca. The observed Ca distribution required that its active transport be postulated, since the membrane was shown to be permeable to Ca and the internal Ca concentration was far below its electrochemical equilibrium value. Metabolic inhibition by iodoacetate or dinitrophenol caused a net Ca uptake, but cooling to 4°C and ouabain did not. Iodoacetate did not affect the Ca45 efflux, but did increase the influx, suggesting that active Ca transport is accomplished by an exclusion mechanism. In experiments with varied external sodium concentrations, no evidence was obtained that sodium competes with calcium for inward transport. Cellular Ca binding was measured under conditions of prolonged metabolic inhibition, which abolished both active transport and the membrane potential. The association constants obtained were compatible with intracellular Ca binding to proteins, but insufficient to account for the low level of intracellular ionic Ca believed essential for relaxation. Hence metabolically dependent intracellular Ca binding was postulated. The Ca45 efflux was slowed down by Ca-free efflux media. The presence of Sr or EDTA could completely prevent this decrease in efflux rate, and Ba could partly prevent it. Changes in Mg and Na concentration did not affect the rate of Ca45 efflux. A model to explain Ca exchange across smooth muscle membranes has been proposed.