We reported previously that astroglia cultured from aquaporin-4-deficient (AQP4^-/-) mice migrate more slowly in vitro than those from wild-type (AQP4^+/+) mice (J. Cell Sci. 2005;118, 5691-5698). Here, we investigate the migration of fluorescently labeled AQP4^+/+ and AQP4^-/- astroglia after implantation into mouse brains in which directional movement was stimulated by a planar stab wound 3 mm away from the axis of the injection needle. Two days after cell injection we determined the location, elongation ratio, and orientation of labeled cells. Migration of AQP4^+/+ but not AQP4^-/- cells toward the stab was greater than away from the stab. AQP4^+/+ astroglia moved on average 1.5 mm toward the stab compared with 0.6 mm for AQP4^-/- cells. More than 25% of the migrating AQP4^+/+ cells but <3% of AQP4^-/- cells appeared elongated (axial ratio>2.5). In transwell assays, AQP4^+/+ astroglia migrated faster than AQP4^-/- cells in a manner dependent on pore size. At 8 h, 50% of AQP4^+/+ cells migrated through 8-µm diameter pores, whereas equivalent migration of AQP4^-/- cells was found for 12-µm diameter pores. These results provide in vivo evidence for AQP4-dependent astroglial migration and suggest that modulation of AQP4 expression or function might alter glial scarring--Auguste, K. I., Jin S., Uchida K., Yan D., Manley G. T., Papadopoulos M. C., Verkman A. S. Greatly impaired migration of implanted aquaporin-4-deficient astroglial cells in mouse brain toward a site of injury.