Stimulation of single Ehrlich ascites tumor cells with agonists (bradykinin, thrombin) and with arachidonic acid (AA) induces increases in the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the presence and absence of extracellular Ca²⁺, measured using the Ca²⁺-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increases in [Ca²⁺]_i, unlike addition of the same agonist twice. Bradykinin and thrombin have additive effects on [Ca²⁺]_i in Ca²⁺-free medium. The phosphoinositidase C inhibitor U-73122 inhibits the agonist-induced increases in [Ca²⁺]_i, whereas ryanodine has no effect. Pretreatment of cells in Ca²⁺-free medium with thapsigargin abolishes the bradykinin-induced increase in [Ca²⁺]_i but not the response to thrombin. The AA-induced response is not inhibited by U-73122 and cannot be mimicked by the inactive structural analog trifluoromethylarachidonyl ketone. Pretreatment of the cells with 50 µM AA (but not with 10 µM AA) abolishes the agonist-induced increase in [Ca²⁺]_i. Thus bradykinin, thrombin, and AA induce increases in [Ca²⁺]_i in Ehrlich cells due to Ca²⁺ entry and release from intracellular stores. Thrombin causes release of Ca²⁺ from an intracellular store that is insensitive to bradykinin and is not depleted by thapsigargin but is depleted by AA.