^<45>Ca-loaded single smooth muscle cells were prepared from taenia coli of guinea pig. The cells were perfused continuously with physiological salt solution on membrane filter with pore size of 12 μm and radioactivity in the effluent was determined. The ⁴⁵Ca-efflux curves consisted of two straight lines in semilogarithmic figure representing loosely and tightly bound calcium compartments with half time of 1.87±0.27 and 6.78±0.43min, respectively. The loosely bound calcium was supposed to be localized on plasma membrane. Size of the tightly bound ⁴⁵Ca was dependent on period for calcium-depletion of the preparation prior to ⁴⁵Ca-loading. The efflux was stimulated by acetylcholine and the stimulated efflux returned to the spontaneous efflux level instantly after removal of acetylcholine. This stimulation was abolished by atropine. Repetitive stimulation failed to increase ⁴⁵Ca-efflux while the rate of the spontaneous ⁴⁵Ca-efflux did not change significantly. These results suggested that, in taenia coli of guinea pig, at least three calcium compartments existed, loosely bound calcium, tightly bound calcium and more tightly bound calcium which can be released by acetylcholine, in addition to calcium in extracellular fluid. Advantages and disadvantages of using single smooth muscle cells for investigation of calcium movement and some technical improvement for determination of ⁴⁵Ca-efflux from single cells were described.