Caspases are implicated in apoptotic cell death after spinal cord injury (SCI), but the relative contribution of these proteases to the secondary injury process has been only partially described. We examined the activation of caspases 1, 2, 3, 6, 8, and 9 from 1 hr to 7 days after moderate contusion injury induced by a weight-drop method in the rat. Tissue homogenates from a 1-cm segment of cord that contained the site of impact were processed by fluorometric enzymatic activity assays and/or immunoblotting methods. Caspases 3, 8, and 9 were activated from 1 to 72 hr after injury, whereas caspases 1, 2, and 6 were not. Double-label immunohistochemistry utilizing antibodies for CNS cell-type-specific markers and active subunits of caspases 3, 8, or 9 showed that, at 4 and 72 hr after injury, these caspases were primarily activated in neurons and oligodendrocytes, rather than in astrocytes. Active caspase subunits were present in neurons within the necrotic lesion core at 4 hr after injury and in cells more than several segments away at 4 or 72 hr after injury. Intrathecal injection of the pan-caspase inhibitor Boc-Asp (OMe)-fluoromethylketone (Boc-d-fmk) at 15 min after injury improved locomotor function 21 and 28 days later. Treatment with the selective caspase 3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD-fmk) improved function at 21 days after injury. These data suggest that caspases 3, 8, and 9 may be differentially activated in white and gray matter after spinal cord trauma and that such activation may contribute to subsequent neurological dysfunction.