The paired-like homeodomain transcription factor CRX (cone-rod homeobox) is involved in regulating photoreceptor gene expression and rod outer segment development. Mutations in CRX have been associated with several retinal degenerative diseases. These conditions range from Leber congenital amaurosis (a severe cone and rod degeneration of childhood onset) to adult onset cone-rod dystrophy and retinitis pigmentosa (an adult onset condition that primarily affects rods). The goal of this study is to better understand the molecular basis of CRX function and to provide insight into how mutations in CRX cause such a variety of clinical phenotypes. We performed deletion analysis in conjunction with DNA binding and transient transfection-based transactivation studies to identify the functional domains within CRX. DNA binding requires a complete homeodomain. Furthermore, truncated proteins that did not contain an intact homeodomain failed to demonstrate detectable expression in tissue culture upon transfection. Transactivation analysis indicated that both the OTX tail and the WSP domain are important for controlling positive regulatory activity of CRX. Interestingly, the mapped CRX transactivation domains were also critical when coexpressed with NRL. Specifically, the synergy between CRX and NRL was constant regardless of which CRX variant was used.