Primary cultures of Müller cells have proven useful in cell biologic, developmental, and electrophysiological studies of Müller cells. However, the limited lifetime of the primary cultures and contamination from non-neural cells have restricted the utility of these cultures. The aim of this study was to obtain an immortalized cell line that exhibits characteristics of Müller cells. Primary Müller cell cultures were prepared from retinas of rats exposed to 2 weeks of constant light. Cells were immortalized by transfection with simian virus 40. Single clones were obtained by repeatedly passaging cells using cloning wells. Immunocytochemical and immunoblotting studies were carried out with glial fibrillary acidic protein (GFAP)-specific and cellular retinaldehyde-binding protein (CRALBP)-specific antibodies. Transient transfections with CRALBP-luciferase constructs were performed by electroporation. Oncogene transformation resulted in the establishment of a permanent cell line that could be readily propagated. Immunocytochemical and immunoblotting studies demonstrated that the Müller cell line, rMC-1, expressed both GFAP, a marker for reactive gliosis in Müller cells, and CRALBP, a marker for Müller cells in the adult retina. Transient transfection assays showed that promoter-proximal sequences of the CRALBP gene were able to stimulate reporter gene expression in rMC-1. Viral oncogene transformation has been successfully used to isolate a permanent cell line that expresses Müller cell phenotype. The rMC-1 cells continue to express both induced and basal markers found in primary Müller cell cultures as well as in the retina. The availability of rMC-1 should facilitate gene expression studies in Müller cells and improve our understanding of Müller cell-neuron interactions.