PURPOSE: The infiltration of inflammatory cells into the cornea is a major determinant in the outcome of keratitis. The purpose of this study was to use enhanced green fluorescence protein (EGFP) bone marrow chimeric mice to visualize and characterize the inflammatory cells that migrate to the corneal stroma during endotoxin-induced keratitis and to explore the mechanisms underlying this process. METHODS: Keratitis was induced by injecting endotoxin into the corneas of EGFP chimeric mice. In vivo fluorescence stereomicroscopy was used to visualize in real time the recruitment of EGFP-positive cells at different time points. Immunohistochemistry and three-dimensional (3D) confocal analysis of whole-mount corneas was used for histologic characterization. Macrophage inflammatory protein-2 (MIP-2) chemokine was neutralized in vivo to determine its contribution to this process. RESULTS: Recruitment of EGFP-positive inflammatory cells in the corneal stroma can be detected in vivo by 6 hours after the injection of endotoxin, and these were mainly neutrophils. Full-thickness whole corneal mount confocal image analysis showed a distinct pattern of migration of EGFP inflammatory cells through the anterior corneal stroma. Moreover, inflammatory cells did not colocalize with the injected lipopolysaccharide (LPS) deposits in the stroma but moved from all directions toward LPS, partially in response to the production of the chemokine MIP-2. CONCLUSIONS: EGFP chimeric mice and ex vivo 3D analysis of whole-mount corneas provides unique information on the interaction of infiltrating inflammatory cells in the cornea. These findings demonstrate that a chemotactic gradient triggered in part by MIP-2 is responsible for directing inflammatory cell migration through the corneal stroma.