Müller cells have been implicated in the pathogenesis of proliferative vitreoretinopathy and subretinal scar formation; however, the source(s) and signal(s) responsible for their activation are unknown. This study was undertaken to determine if the retinal pigment epithelium (RPE) could be involved in this signaling process by studying its effects on Müller cell survival and division in vitro. A pure population of Müller cells isolated from 1-2 day Long-Evans rats was seeded at low density and treated with medium conditioned by neonatal rat RPE (RPE-CM) or a nonconditioned, defined medium. By day 3, Müller cells cultured in RPE-CM increased in number 2-fold. These cells survived up to 21 days, which was the longest time tested. In contrast, cell number decreased in control wells 75% by day 3, and 100% by day 4. The RPE-mediated survival and proliferation of the Müller cells occurred in a dose-dependent manner. The mitogenic response was specific for the RPE when compared with fibroblasts and non-retinal epithelial cells. Heat and trypsin treatment of the RPE-CM completely abolished its survival and mitogenic activity. These findings demonstrate the establishment of an in vitro model which can be used to investigate RPE-Müller cell interactions. This study also provides evidence for RPE involvement in Müller cell interactions. This study also provides evidence for RPE involvement in Müller cell survival and proliferation.