Retinoid trafficking between the photoreceptors and pigmented epithelium is probably mediated by interphotoreceptor retinoid-binding protein (IRBP), a 124-145 kDa glycolipoprotein in mammals and amphibians. In these animals, IRBP is composed of four homologous regions (modules) 300 amino acids in length. We have determined the primary structure of zebrafish IRBP and its expression pattern by northern analysis, reverse transcriptase-polymerase chain reaction and in situ hybridization under a variety of lighting conditions. Zebrafish IRBP is half the size (66.3 kDa) of mammalian IRBP because it is composed of only two modules, similar to goldfish IRBP. The first half of the zebrafish protein is most similar to the first module of mammalian IRBP and the second half to the fourth module of mammalian IRBP. This suggests that during the evolution of the ray-finned fish (Actinopterygii), the middle two modules were lost. Each of the modules contains conserved hydrophobic domains which may form the ligand-binding pocket. The expression of zebrafish IRBP mRNA is sevenfold higher in the middle of the light period (at mid-light) than in the middle of the dark period (at mid-dark). This rhythm persists for 2 days under conditions of constant light or constant darkness, then dampens to an intermediate level by 8 days of constant conditions. At mid-light, IRBP mRNA is expressed by all cone types and to a lesser extent by the rods. At mid-dark, the mRNA is restricted to the ultraviolet-sensitive short single cones. These data suggest that IRBP expression is regulated by circadian and light-driven mechanisms that act differentially on the various photoreceptor subtypes in the zebrafish retina.