The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined. Copyright © 2012 by the American Association of Neuropathologists, Inc.