Calcium ions (Ca2+) are accumulated by a cellular depot in isolated rabbit ileal longitudinal smooth muscle during incubation of this tissue in high Ca2+ medium. Transfer of the muscle to Ca2+-free solution results in a transient contractile response. The magnitude of the contraction appears to be a rectangular hyperbolic function of the Ca2+ concentration in the cellular compartment. When 10−6M acetylcholine (ACh) is present in the Ca2+-free solution, the muscle is stimulated, and the magnitude and duration of the mechanical activity occurring in the Ca2+-free environment is enhanced. When 10−6 or 10−7M epinephrine (Epi) is present in the Ca2+-free medium, the magnitude and duration of the contraction occurring in the Ca2+-free solution is reduced. Neither agent alters significantly the rate of loss of radioactive 45Ca from the muscle strips. Rather, these agents may be acting by influencing the operation of a saturable Ca2+ transport system located between the cellular Ca2+ pool and the contractile machinery. Evidence indicates that ACh increases and Epi decreases the turnover rate and/or number of active Ca2+ sites associated with this transport mechanism.