Established cell lines are now widely used in experiments concerning vascular smooth muscle (VSM) function; however, considerable evidence suggests that cultured VSM cells are functionally different from VSM cells in intact blood vessels. In order to test the hypothesis that calcium signaling mechanisms are comparable in these two preparations, we developed a new method for high resolution 45Ca efflux studies in A7r5 cells. Briefly, this method involves plating cells in the lumen of a tubular glass efflux chamber and, after loading the cells with 45Ca, perfusing the chamber with a physiological saline solution and collecting the effluent. Using this method we found that the plasma membrane in cultured cells is not rate limiting for calcium efflux, since the efflux curves from both permeabilized and intact cells are kinetically the same. We also found the plasma membrane is not rate limiting in whole aortic segments by using a depolarizing solution followed by dihydropyridine solution. Thus, we demonstrated that the data obtained from cells or tissues with intact membranes reveal information about the intracellular stores (sarcoplasmic reticulum and mitochondria). Combining efflux data with a detailed kinetic model of cellular Ca transport allows least-squares estimation of the rate constants for release and uptake of Ca2+ by intracellular stores with a high degree of confidence (CV < 25%) as well as the Ca2+ contents and transmembrane fluxes associated with these stores. Quantitative comparison of results obtained from A7r5 cells with those we previously obtained for rabbit aortic segments reveals marked similarities and suggests that A7r5 cells serve as excellent model experiments for VSM cell Ca2+ homeostasis.