Calcium exchange was measured in enzymatically isolated, cultured smooth muscle cells from the rabbit thoracic aorta. Cells were grown to confluence in monolayer on scintillator discs which were then inserted into a modified flow cell/spectrometer to measure 45Ca exchange in a continuous, on-line manner. This also allowed us to measure rapid movements of cellular Ca2+ without solubilizing the cells. Two components of Ca2+ exchange were detected: a La3+-displaceable, rapidly exchangeable fraction (t 1/2 = 15.6 s) and a slowly exchangeable fraction (t 1/2 = 34.5 min). The La3+ displaceable, rapidly exchangeable Ca2+ fraction represented 57 to 61% of the total exchangeable Ca2+. Multiple passaging of cells did not after the Ca2+ flux characteristics. Low Na+ perfusion increased smooth muscle cell Ca2+ flux by 6.71 mmol/kg dry weight. This increase was localized to both the rapidly and slowly exchangeable Ca2+ compartments. Ouabain, an inhibitor of the plasmalemmal Na+ -K+ pump, also increased net uptake of 45Ca. The results demonstrate that approximately 60% of exchangeable Ca2+ of smooth muscle cells is very rapidly exchangeable (t 1/2 less than 16 s). The results stress the importance of measuring 45Ca2+ movements in an on-line, continuous manner in order to ensure detection of the majority of total exchangeable Ca2+ in smooth muscle cells.