The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl-/- rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2 and RORβ, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2 and RORβ can bind to the critical region in vitro, while chromatin immunoprecipitation (ChIP) experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2 and RORβ directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORβ and later maintained at high levels by CRX and RORβ.