Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuronal cells. However, the integrated ES cells also have a tumorigenic effect just because they have the ability for multipotential differentiation to various types of tissues. Thus, control of neoplastic potentials of ES cells is very important for the treatment of degenerative or injured diseases. Mouse ES cells carrying the sequence for the green fluorescent protein (GFP) gene were transplanted into adult mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. One week after the ES cell injection, folate antagonist methotrexate (MTX) was injected intravitreally. Eyes were retrieved 4 weeks after ES cell transplantation for histologic analyses. Conventional histological analysis was performed by hematoxylin and eosin staining with the use of paraffin-embedded sections. Neuronal differentiation and teratogenic potential of ES cells were demonstrated by immunohistochemistry. The proliferative activity of transplanted cells was detected by mitotic index, proliferating cell nuclear antigen index and AgNOR count. The incorporation of transplanted ES cells in MTX-treated and non-treated retinas at 4 weeks after transplantation was observed in 8/16 eyes (50%) and 8/16 eyes (50%), respectively. Transplanted ES cells in MTX-treated retina showed increased neuronal differentiation and decreased expression of teratogenic markers, compared with ES cells in non-treated retina. The proliferative activity of transplanted ES cells in MTX-treated retina was lower than that in non-treated retina. These results suggest that intravitreal MTX treatment following transplantation can induce neuronal differentiation in the transplanted ES cells and decrease their proliferative activity.