In the nervous system serine proteases, like thrombin, are involved in developmental and repair processes, but serve also as extracellular signalling molecules, acting via protease-activated receptors. Cellular responses of glial cells to thrombin are transduced by proteolytic activation of the G protein-coupled thrombin receptor. A second member of the protease-activated receptor family, protease-activated receptor-2, is activated by trypsin. We assessed whether glial cells express protease-activated receptor-2 together with the thrombin receptor. By reverse transcriptase polymerase chain reaction and Ca2+ imaging studies we demonstrate that rat astrocytes and C6 glioma cells functionally express protease-activated receptor-2. Short-term stimulation of the glial cells with thrombin, thrombin receptor agonist peptide, trypsin and protease-activated receptor-2 activating peptide dose-dependently induced a transient rise of [Ca2+]i. In astrocytes omission of extracellular Ca2+ attenuated the amplitude of the [Ca2+]i transient induced by protease-activated receptor-stimulation. The decrease was strongest for the trypsin-evoked response and a reduction comparable in size (40%) was observed by pre-treatment with pertussis toxin. In astrocytes concentration-effect curves reveal that (i) the proteases had a higher potency than the respective receptor-activating peptides to induce a Ca2+ response, (ii) proteolytic activation of the receptors by thrombin or trypsin resulted in a double-sigmoidal concentration-effect curve, whereas non-proteolytic activation by receptor activating peptides resulted in a sigmoidal concentration dependence, and (iii) trypsin evoked a significantly greater Ca2+ response than thrombin. Preceding stimulation with trypsin nearly abolished the subsequent response to thrombin, whereas the trypsin-evoked Ca2+ transient was only slightly attenuated after a prior challenge with thrombin. This is the first study to show that neural cells (glial cells) functionally express both thrombin receptor and protease-activated receptor-2 coupled to the mobilization of intracellular calcium. Since calcium is the premier second messenger mediating adaptive changes within the CNS, these findings emphasize an important physiological function of serine proteases in mammalian brain.