Endothelins are a family of conserved vasoactive peptides that are widely expressed in different biological systems including the eye. In the cell culture model of retinal pigment epithelium, ARPE-19, the synthesis and secretion of endothelin-1 (ET-1) is regulated by cholinergics and TNF-α. In the present study we investigated the expression of ET-1 in RPE in situ, in rat and human eyes. Additionally, we have employed the human retinal pigment epithelial (ARPE-19) cells to delineate the apical and basolateral ET-1 expression and secretion by confocal microscopy and radioimmunoassay, respectively. Our results suggest a possible conservation of ET-1 expression predominantly in the mammalian RPE underlining its importance at this site. Additionally, our results suggest that constitutive ET-1 secretion is predominantly towards the basolateral side in cultured RPE possibly allowing ET-1 to activate its receptors located in the choroidal blood vessels and regulate retinal and choroidal blood flow.