The fluorescence and secretory response of calcium-chlorotetracycline were simultaneously and continuously measured in isolated perfused rat pancreata. Continuous vascular perfusion with 10 μM chlorotetracycline increased the fluorescence intensity exponentially. Continuous stimulation with higher nonphysiological doses of CCK (20 mU/ml) or ACh (10−6M induced (a) a gradual decrease in the Cacholorotetracycline fluorescence which increased to control after cessation of the stimulation, and (b) secretory response, consisting of an initial rapid ascent followed by a decline. On the other hand, continuous stimulation with a lower concentration of CCK (5 mUim1) or ACh (5 × 10−8M), which seems to be rather physiological, did not change the time course of the Ca-chlorotetracycline fluorescence increase, but it evoked a definite secretory response, consisting of an initial rapid ascent to a maximum followed by a descent to a sustained plateau. In a Ca2+-deficient environment, stimulation with 5 mU CCK/ml evoked only a small secretory response and a gradual insignificant decrease in the Ca-chlorotetracycline fluorescence. The present results may support the view that continuous stimulation with secretagogues at physiological concentrations does not cause dissociation of stored Ca2+from intracellular sites but does initiate secretion probably via the rise in [Ca2+], induced by the influx of extracellular Ca2+.