This study concerns changes of intracellular calcium concentrations, [Ca2+]i, in human umbilical vein endothelial cells (HUVECs) in response to thrombin, platelet activating factor (PAF), and leukotriene B4 (LTB4). Thrombin (0.003 to 1 U/ml) induced a dose-dependent, pertussis toxin-insensitive rise of [Ca2+]i that was initially rapid and transient and was followed by a sustained plateau phase that required extracellular calcium to be expressed. Early during that plateau a second rise of [Ca2+]i was seen that was amplified in the presence of propranolol and abolished in calcium-free medium or by ethanol. Repeated stimulations with thrombin evoked renewed but declining responses. Pretreatment of HUVECs with PAF or lipoxin A4, but not LTB4, diminished a subsequent response to thrombin. PAF induced a small, dose-dependent increase of [Ca2+]i with kinetics similar to that of thrombin. It was blocked by previous exposure of HUVECs to PAF and thrombin but not to LTB4 or pertussis toxin. LTB4 induced a rapid but sustained increase of [Ca2+]i that resembled that of the calcium ionophore ionomycin. In contrast to responses to thrombin and PAF, it could be abolished by previous treatment with pertussis toxin and required extracellular calcium. Addition of LTB4 after previous stimulation with LTB4 or PAF gave no detectable response. Implications of these results for some specific signal transduction mechanisms for agonists studied are discussed.