Mechanical spinal cord injury (SCI) initiates a cascade of pathochemical and pathophysiological events, collectively known as the secondary injury. There has been a long-standing interest in understanding the activation and involvement of proteases in this secondary injury process. Several proteases including the calpains, caspases and matrix metalloproteinases are activated by perturbations to the spinal cord and have been linked to cell death following SCI and in other models of CNS disease and insult. Cathepsin B (Cath B), a potent lysosomal protease, has also been implicated in the pathology of CNS diseases including brain tumors, Alzheimer's disease, amyotrophic lateral sclerosis and stroke. Previously, we reported significant increases in Cath B mRNA and protein expression following contusion-SCI. This characterization of Cath B continues with the experiments reported herein, which were designed to examine Cath B enzymatic activity and cellular localization following contusion-SCI in the rat. Cath B enzymatic activity was significantly increased in the injury epicenter at 5 and 7 days post-injury and was highly correlated with increases in the active forms of the Cath B protein reported earlier. Furthermore, the immunohistochemical analyses revealed that the post-injury increases in expression and enzymatic activity at the injury epicenter were due to the presence of a large and diverse population of inflammatory cells. However, in areas adjacent to the injury epicenter, it appears that parenchymal neurons may also contribute to these increases. Our findings coupled with the documented role of Cath B in other CNS pathologies make this potent protease an attractive candidate for involvement in the tissue destruction associated with the secondary injury cascade following SCI.