Microglial cells (MCs) are active sensors and reactive phagocytes of neural tissues. They are known to migrate and accumulate in areas of neuronal damage. Thus, microglial locomotion is an essential feature of the inflammatory reaction in neural tissue. Yet, to our knowledge there has been no report of direct in vivo observation of the migration of MCs. Here, we show that intravitreally injected cyanine dyes (DiO, DiI, and indocyanine green) are sequestrated in MCs during several months, and subsequently in vivo images of these fluorescent MCs can be obtained by confocal scanning laser ophthalmoscopy. This enabled noninvasive, time-lapse observation of the migrating behavior of MCs, both in the basal state and following laser damage. In the basal state, a slow, intermittent, random-like locomotion was observed. Following focal laser damage, MCs promptly (i.e., within 1 h) initiated centripetal, convergent migration. MCs up to 400 μm away migrated into the scar at velocities up to 7 μm/min. This early phase of centripetal migration was followed by a more prolonged phase of nontargeted locomotion around and within injured sites during at least 24 h. Cyanine-positive cells persisted within the scar during several weeks. To our knowledge, this is the first in vivo observation of the locomotion of individual MCs. Our results show that the locomotion of MCs is not limited to translocation to acutely damaged area, but may also be observed in the basal state and after completion of the recruitment of MCs into scars.