Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause X-linked retinitis pigmentosa type 3 (RP3), a severe, progressive and degenerative retinal dystrophy eventually leading to complete blindness. RPGR is ubiquitously expressed, yet mutations in the RPGR gene lead to a retina-restricted phenotype. To date, all RP3 associated missense mutations that have been identified are located in the RCC1-homologous domain (RHD) of RPGR. To investigate the molecular pathogenesis of RP3, we screened retinal yeast two-hybrid libraries with the RHD of RPGR. We identified several alternatively spliced gene products, some with retina-restricted expression, that interact specifically with RPGR in vivo and in vitro. Thus, these proteins were named RPGR-interacting protein 1 (RPGRIP1) isoforms. They contain a C-terminal RPGR-interacting domain and stretches of variable coiled-coil domains homologous to proteins involved in vesicular trafficking. The interaction between RPGR and RPGRIP1 isoforms was impaired in vivo by RP3-associated mutations in RPGR. Moreover, RPGR and RPGRIP1 co-localize in the outer segment of rod photoreceptors, which is in full agreement with the retinitis pigmentosa phenotype observed in RP3 patients. The localization of RPGRIP1 at 14q11 makes it a strong candidate gene for RP16. These results provide a clue for the retina-specific pathogenesis in RP3, and hint towards the involvement of RPGR and RPGRIP1 in mediating vesicular transport-associated processes.