Thrombin, a serine protease essential for blood coagulation, also plays an important role in cellular injury associated with intracerebral hemorrhage. Here, we show that, in organotypic cortico-striatal slice cultures, thrombin evoked delayed neuronal injury in the cerebral cortex and shrinkage of the striatum. These effects were prevented by cycloheximide and actinomycin D but not by a caspase-3 inhibitor. Thrombin-induced shrinkage of the striatum was abolished by a thrombin inhibitor argatroban or prior heat inactivation of thrombin, and significantly attenuated by a protease-activated receptor-1 antagonist FR171113. However, thrombin-induced cortical injury was not prevented either by heat inactivation or by FR171113, and was only partially inhibited by argatroban. In addition, inhibition of extracelluar signal-regulated kinase (ERK), Src tyrosine kinase and protein kinase C prevented both neuronal injury in the cortex and shrinkage of the striatum, whereas inhibition of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase prevented shrinkage of the striatum only. Thrombin treatment promptly induced phosphorylation of ERK, which was not prevented by inhibition of Src and protein kinase C. Thus, thrombin induces cellular injury in the cerebral cortex and the striatum, by recruiting multiple and distinct signaling pathways in protease activity-independent as well as dependent manner.