This study aimed to establish an stable in vitro culture model for rat retinal ganglion cells (RGCs). Retinal cells were isolated from 1-3 days old Sprague-Dawley (SD) rats. Cell suspension was made by digesting the retina tissues with 0.2% trypsin. The cells were seeded in a plate, and grown in 37 degrees C, 5% CO2 with a seeding density of 2.0 x 10(6) ml(-1). One-third of cell culture media was replaced and cytosine arabinoside was added (with a final concentration of 10 mu mol L-1) at the 72 hr to inhibit the growth of contaminated cells. Fluorescence labeled Thy1.1 and DAPI stain were used to identify RGCs and total cells, respectively. Evenly distributed cells in scaffolds were observed under microscope after seeding. The cells appeared normal with growing axons and partial neurite elongation during the seven days of culture. After 96 hr of seeding, typical RGCs were shown as dark blue triangle with their axonal projections and a few bigger glial cells were seen in the background. Specific antibody Thy1.1 stain and DAPI nucleic acid stain showed that 92.3% of the cells were RGCs. Thus, in vitro RGC culture model was established successfully.