We have used indo-1-loaded human endothelial cells (EC) in monolayer culture and quantitative laser scanning fluorescence microscopy techniques to investigate the magnitude and duration of the change in cytoplasmic free calcium ([Ca2+]i) required for thrombin-stimulated von Willebrand factor (vWF) secretion in individual EC. Both alpha-thrombin and a 14 amino acid thrombin receptor activating peptide stimulate an increase in EC [Ca2+]i that is agonist dose dependent. Low-dose agonist treatment generates asynchronous oscillations (i.e., repetitive spikes < 80 sec duration) in [Ca2+]i. Stimulation with higher agonist concentrations generates a prolonged single peak elevation in [Ca2+]i. Both the number of cells displaying prolonged [Ca2+]i peaks and the mean amplitude of the peaks increase as a function of agonist concentration. Higher doses of agonist also cause sustained elevations in [Ca2+]i that depend upon extracellular Ca2+. Oscillations in [Ca2+]i are not sufficient to stimulate significant vWF secretion, and sustained elevations in [Ca2+]i are not required for maximal secretion. Both the number of cells displaying prolonged peaks and the mean peak amplitude correlate with increasing levels of vWF secretion from the culture. We have used the expression of P-selectin, a secretory granule membrane protein, as a marker for measuring thrombin-induced exocytosis in individual EC. Both the number of secreting cells and the amount of secretion per cell increase as a function of thrombin concentration. The graded responses in [Ca2+]i amplitudes and the graded exocytotic response may be causally related.