The X-linked RP3 locus codes for retinitis pigmentosa GTPase regulator (RPGR), a protein of unknown function with sequence homology to the guanine nucleotide exchange factor for Ran GTPase. We created an RPGR-deficient murine model by gene knockout. In the mutant mice, cone photoreceptors exhibit ectopic localization of cone opsins in the cell body and synapses and rod photoreceptors have a reduced level of rhodopsin. Subsequently, both cone and rod photoreceptors degenerate. RPGR was found normally localized to the connecting cilia of rod and cone photoreceptors. These data point to a role for RPGR in maintaining the polarized protein distribution across the connecting cilium by facilitating directional transport or restricting redistribution. The function of RPGR is essential for the long-term maintenance of photoreceptor viability.R etinitis pigmentosa (RP) is a group of hereditary retinal diseases in which photoreceptor cells degenerate. The genetic defects underlying RP are heterogeneous, with well documented autosomal and X-linked forms (http:͞͞www.sph.uth. tmc.edu͞RetNet). Many of the genes involved code for well characterized functions essential for the rod phototransduction cascade or for the maintenance of the photoreceptor outer segment structure. Others encode unknown functions. Among the latter is the X-linked locus RP3 (OMIM 312610), which accounts for the majority of X-linked RP families by linkage studies. The retinal disease associated with RP3 appears to have an early age of onset (1) and exhibits early involvement of both cones and rods (2-7). This is in contrast with other types of RP in which rods are primarily affected followed by a secondary degeneration of cones. Because daytime vision and visual acuity depend on cone function, patients with RP3 would experience visual handicaps earlier and with greater severity than the average RP patients. The clinical expression of RP3 suggests that the genetic defect affects an essential function for both rods and cones.The gene at the RP3 locus was recently isolated and found to encode retinitis pigmentosa GTPase regulator (RPGR) (8, 9), so named because of its similarity to the regulator of chromatin condensation (RCC1), a nuclear protein that catalyzes guanine nucleotide exchange for the small GTPase Ran. The N-terminal half of RPGR consists of six complete tandem repeats of 52-54 amino acids with homology to the repeat structure of RCC1. RCC1 has an essential role in nuclear import and export through its regulation of Ran (10). The presence of an RCC1 homology domain thus raises the possibility that RPGR might participate in some type of intracellular transport process in photoreceptors. The function of RPGR remains unknown (11,12). To investigate the in vivo function of RPGR and to understand the disease mechanism associated with RPGR mutations, we generated an RPGR-deficient mouse model and examined its phenotype. We also determined the normal subcellular localization of RPGR in photoreceptors. Our results are consistent with RPGR being involved...