PURPOSE. To evaluate a novel 3D culture model of the corneal stroma and apply it to investigate how key wound-healing growth factors regulate the mechanics of corneal keratocyte migration. METHODS. Rabbit corneal keratocytes were seeded within collagen matrices that were compacted using external compression. Six-millimeter-diameter buttons were then incubated in media supplemented with 10% FBS, TGFβ1, TGFβ2, plateletderived growth factor (PDGF), or no growth factor (control). After 1, 3, or 7 days, matrices were labeled with phalloidin and a nucleic acid dye, and were imaged using laser confocal microscopy. To study cell migration, buttons were nested within acellular uncompressed outer collagen matrices before growth factor stimulation. RESULTS. Corneal keratocytes in basal media within compressed matrices had a broad, convoluted cell body and thin dendritic processes. In contrast, cells in 10% FBS developed a bipolar fibroblastic morphology. Treatment with TGFβ induced the formation of stress fibers expressing α-smooth muscle actin, suggesting myofibroblast transformation. PDGF induced keratocyte elongation without inducing stress fiber formation. Both 10% FBS and PDGF stimulated significant keratocyte migration through the uncompressed outer matrix, but 10% FBS produced more cell-induced collagen matrix reorganization. TGFβ induced the smallest increase in migration and the greatest matrix reorganization. CONCLUSIONS. Corneal keratocytes are able to differentiate normally and respond to growth factors within compressed collagen matrices, which provide a high-stiffness, 3D environment, similar to native stromal tissue. In addition, nesting these matrices provides a unique platform for investigating the mechanics of keratocyte migration after exposure to specific woundhealing cytokines.