The rapid loss of neurons is a major pathological outcome of intracerebral hemorrhage (ICH). Several mechanisms may produce the neurotoxicity observed in ICH, and these include proteolytic enzymes such as thrombin and matrix metalloproteinase-9 (MMP-9). We tested the hypothesis that thrombin and MMP-9 combine to injure neurons in culture and that they interact to promote the acute neurotoxicity that occurs in ICH in vivo. We report that human fetal neurons die when exposed to thrombin or MMP-9 in isolation and that a combination of these two enzymes increased neurotoxicity. The toxicity of thrombin involved protease-activated receptor-1 and the conversion of proMMP-9 to active MMP-9. In ICH, which was induced in mice by the intracerebral injection of autologous blood, significant areas of brain damage, neuronal death, microglia/macrophage activation, and neutrophil accumulation occurred by 24 h of injury. Importantly, these neuropathological features were reduced in MMP-9 null mice compared with wild-type controls, and the concordant antagonism of thrombin using hirudin also alleviated the injury found in MMP-9 null mice. Our collective results demonstrate that thrombin and MMP-9 collaborate to promote neuronal death in culture and in ICH. To improve the prognosis of ICH, the neurotoxic actions of thrombin and MMP-9 must be inhibited early and simultaneously after injury.