PURPOSE: To characterize the timing of differentiation, antigenic expression, morphology, proliferative potential, and apoptosis during astrocyte differentiation in the rat retina in vivo. METHODS: Whole mounts of rat retinae from embryonic day (E) 13 to postnatal day (P) 21 and adults were examined utilizing combinations of Pax2, GFAP, vimentin, S100, and GS lectin. These markers were also combined with BrdU and TUNEL to identify proliferation and apoptosis of cells of the astrocytic lineage. RESULTS: Three distinct stages of astrocytic differentiation were identified: (i) Pax2+/vimentin+/GFAP(-) astrocyte precursor cell (APC), (ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes, and (iii) Pax2+/vimentin(-)/GFAP+ mature perinatal astrocytes. An earlier transient site of astrocyte generation was detected from E13 to E15 at the ventricular surface, but unlike the majority of retinal astrocytes that migrate into the retina starting at E15-E16, this ventricular source of retinal astrocytes were restricted to a small rim surrounding the optic nerve head. APCs and perinatal astrocytes were highly proliferative and migratory. Significant numbers of perinatal astrocytes were lost because of apoptosis, which was matched closely to the retraction of excess capillary segments during postnatal maturation of the retinal vasculature. CONCLUSIONS: This study provides evidence of a second site of astrocyte generation at the ventricular zone early in embryonic development of the mammalian retinae. APCs are present from E16 to E20 only during perinatal development and are a highly migratory and proliferative cell. As the retina is considered a part of the central nervous system (CNS), this is the first in vivo characterization of cells of the astrocytic lineage in mammalian CNS development.