The protease thrombin seems to play a central role in events following neural injury, whereby the enzyme can act, in concert with other molecules as a hormone or as a growth factor. In cells derived from the nervous system, thrombin induces changes in morphology and proliferation. The signalling mechanisms involved in these thrombinactivated processes are still unclear. In the present study we investigated Ca 2ϩ signals in fura-2 loaded rat astrocytes in primary culture. Brief stimulation of astrocytes with thrombin induced a dose-dependent transient elevation of [Ca 2ϩ ] i , best fitted by a double-sigmoidal curve giving two EC 50 values of 3 pM and 150 pM. Continuous superfusion of cells with thrombin induced Ca 2ϩ responses with three different types of kinetics. In 48% of the cells tested a single transient rise superimposed with fast fluctuations of [Ca 2ϩ ] i was seen. The following complex long-term changes of [Ca 2ϩ ] i , dependent on the presence of the agonist thrombin, were observed: i) a biphasic [Ca 2ϩ ] i elevation, characterized by an initial peak followed by a sustained plateau phase (in 43% of the cells) and ii) oscillations of [Ca 2ϩ ] i (in 9% of the cells). The observed Ca 2ϩ responses were inhibited by the phospholipase C (PLC) inhibitor U-73122 and the thrombin inhibitor protease nexin-1/glia-derived nexin. The synthetic thrombin receptor activating peptide could mimic the thrombin-induced changes of [Ca 2ϩ ] i . In astrocytes in Ca 2ϩ -free medium, thrombin induced a sharp single transient Ca 2ϩ rise, without superimposed fluctuations. After depletion of intracellular Ca 2ϩ stores with thapsigargin the Ca 2ϩ response to thrombin was diminished or completely suppressed indicating that thrombin induces the release of Ca 2ϩ from intracellular stores. During long-term Ca 2ϩ responses, omission of extracellular Ca 2ϩ resulted in a reversible interruption of the signal. In conclusion our results demonstrate that thrombin by activation of its plasma membrane receptor induces through activation of PLC different types of Ca 2ϩ responses. The complex Ca 2ϩ signals are generated by an interplay of InsP 3 -mediated Ca 2ϩ release from intracellular stores and Ca 2ϩ entry across the plasma membrane.