Sphingosine-1-phosphate (S1P) is a pleiotropic lysolipid that has recently been implicated in the regulation of tissue fibrosis. However, the fibrogenic potential of S1P in the eye has not previously been investigated. In the current study, we evaluated cells from the anterior and posterior segments of the eye for the presence of S1P and their potential ability to produce and respond to S1P. In addition, we investigated the regulatory role of S1P as a mediator of proliferation, cellular transformation and pro-fibrotic protein expression in human retinal pigmented epithelial cells. Expression of S1P receptors and sphingosine kinases (the enzymes that produce S1P) was examined using RT-PCR, and intracellular localization of S1P was examined using immunoblotting, immunohistochemistry and ELISA in primary human retinal pigmented epithelial (RPE) cells, primary human conjunctival fibroblasts (ConF), and primary human corneal fibroblasts (CF). RPE cell proliferation was determined using an MTT-based cell proliferation assay, and RPE myofibroblast transformation, collagen type I production and profibrotic protein expression were assessed using immunofluorescence, ELISA and immunoblot. S1P(1-3, 5) receptors and sphingosine kinases 1 and 2 were expressed and intracellular pools of S1P were detected in RPE cells, ConF and CF. S1P stimulated RPE cell proliferation in a dose- and time-dependent manner. S1P induced myofibroblast transformation of RPE cells, as indicated by increased alpha-smooth muscle actin (alpha-SMA) expression and its incorporation into prominent stress fibers, and promoted collagen type I production. S1P stimulated the expression of plasminogen activator inhibitor-1 (PAI-1) and heat shock protein 47 (HSP47), two proteins that are linked to increased tissue fibrosis. Combined, these data demonstrate that RPE cells, ConF and CF from the human eye not only have the molecular ability to produce and respond to S1P, but also contain S1P. Furthermore, S1P promotes proliferation, myofibroblast transformation, collagen production and pro-fibrotic protein expression by human RPE cells. These data suggest that S1P is a previously unrecognized mediator of profibrotic cellular function and signaling in the eye.