Despite the importance of the retinal pigment epithelium (RPE) for vision, the molecular processes involved in its specification are poorly understood. We identified two new mutant alleles for the zebrafish gene chokh (chk), which display a reduction or absence of the RPE. Unexpectedly, the neural retina (NR) in chk is specified and laminated, indicating that the regulatory network leading to NR development is largely independent of the RPE. Genetic mapping and molecular characterization revealed that chk encodes Rx3. Expression analyses show that otx2 and mitfb are not expressed in the prospective RPE of chk, indicating that the retinal homeobox gene rx3 acts upstream of the molecular network controlling RPE specification. Cellular transplantations demonstrate that rx3 function is autonomously required to specify the prospective RPE. Though rx2 is also absent in chk, neither rx2 nor rx1 is required for RPE development. Thus, our data provide the first indication that, in addition to controlling optic lobe evagination and proliferation, chk/rx3 also determines cellular fate.