Proteases such as matrix metalloproteinases (MMPs) and thrombin are implicated in intracerebral haemorrhage (ICH) but their interactions amongst one another and interdependency remain to be defined. The latter is important since proteases acting through different mechanisms to inflict neurotoxicity would require separate targeting compared with proteases acting through the same cascade. We reported recently that MMP-9 and thrombin combined to promote neurotoxicity in ICH; however, as there was still substantial injury when both MMP-9 and thrombin were inhibited, we sought other factors that also contribute to ICH pathology. MMP-3, another member of the MMP family, has been correlated with poor prognosis in ICH in humans and it has been shown to increase rapidly after ICH in animals. Moreover, MMP-3 can convert the MMP-9 zymogen to its active form. Thus, we have examined whether MMP-3 is neurotoxic and addressed whether its potential effect in ICH is dependent on, or additional to, damage inflicted by MMP-9 and thrombin. We report that cultured neurons are killed by MMP-3 and that neuronal death is most marked when all three proteases, MMP-3, MMP-9 and thrombin, are combined. In vivo, the injection of autologous blood into the right striatum to produce ICH injury resulted in MMP-3 expression within 3 h. The blood-induced lesion and neuronal death was significantly reduced in MMP-3 or MMP-9 null mice compared with wild-type counterparts, and MMP-3 and -9 double null mice had even less brain damage. Significantly, pathological destruction after ICH was least in MMP-3 and -9 double null mice treated with a thrombin antagonist, hirudin. These results provide insights into molecules that inflict neurotoxicity in ICH and demonstrate that multiple proteases would need to be targeted simultaneously to successfully reduce ICH neurotoxicity.