A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.