Activation of Microglia/macrophages has been observed in ischemia-reperfusion injury of the brain. This study was undertaken to investigate the different subpopulations of microglia/macrophages in the normal rat retina and their activation after retinal ischemia. Retinal ischemia was induced by elevation of intraocular pressure to 120 mmHg for 60 min. Microglia/macrophages were identified on frozen retinal sections by four antibodies, namely OX42, 5D4, OX6 and ED1. In the normal retina, there were heterogeneous populations of resident microglia/macrophages as characterized by their differences in morphology, antigen expression and distribution. OX42+ cells had delicate processes and were located in the inner layers of the retina, while 5D4+ cells were highly ramified and mostly scattered in the inner plexiform layer (IPL) and the outer plexiform layer. Few amoeboid ED1+ cells were also seen in the ganglion cell layer and IPL. OX6+ (MHC-II antigen presenting) cells were not detected in the normal retinas. Double labeling with OX42 and 5D4 antibodies on normal retinal sections showed few microglia exhibited positive labeling with both OX42 and 5D4, while the majority of the microglia were labeled with either OX42 or 5D4 antibodies. After retinal ischemia single labeling with these antibodies showed increased number of these antigen-expressing cells, disappearance of normal cellular processes, and rounding or amoeboid like appearance of the cell bodies. At 1 day after ischemia, there was a significant infiltration of round OX42+, ED1+ and OX6+ cells with loss of the cellular processes in the inner retina. From 3 to 14 days, all subpopulations of microglia/macrophages differentiated cellular processes and became dendritic again. Double labeling on retinas after 1 day of recovery showed OX42+ cells were co-labeled with ED1+ or OX6+ cells, but not with 5D4+ cells. Scattered amoeboid OX42+, 5D4+, and ED1+ cells were noted in the subretinal space 3-14 days after ischemia. In summary, there were heterogeneous populations of resident microglia/macrophages in the normal inner retina and they were activated early after ischemia-reperfusion injury and exhibited different antigenic expression which were further altered in the recovery phase.