OBJECTIVE We wished to isolate and cultivate adult human retinal ganglion cells (RGC) from donor eyes. METHODS Small pieces of retina from donor eyes were plated in dishes and cultured with Ham's F12 medium with 10% serum for organ culture. For cell culture, cells were isolated by mechanical or enzymatic dissociation methods and cultured with F12 medium with 10% serum, with or without nerve growth factor (NGF) and/or basic fibroblast growth factor (bFGF). RESULTS In organ cultures, no neurite outgrowth from the retinal explants was observed. In cell cultures for which mechanical dissociation methods were used, the few cells that could be isolated showed poor viability. Better results were obtained with enzymatic dissociation methods. When cultured with medium supplemented with bFGF, some cells attached, spread, and sent out numerous dendrites, morphologically similar to RGC. These cells stained positively for neurofilaments and Thy-1 and negatively for glial fibrillary acidic protein (GFAP), indicating they were RGC. CONCLUSIONS Cell cultures of human RGC can be established. This is a potential model system for studying effects of damaging and protective factors on RGC in vitro.