We recently identified a novel retinoid binding protein, Papilio RBP, in the soluble fraction of the eye homogenate of the butterfly Papilio xuthus, and demonstrated that the protein is involved in the visual cycle. We now have localized the protein in the Papilio eye by light and electron microscopic immunohistochemistry using a monospecific antiserum produced against artificially expressed Papilio RBP. We found strong immunoreactivity in the primary as well as secondary pigment cells and in the tracheal cells. The pigment cells have long been regarded as an important site of the visual cycle, and this view is further supported by the present result. Interestingly, the cytoplasm and nuclei of these cells were equally labeled, indicating that the protein exists in both the cytoplasm and the nucleus. We conducted a survey for the existence of the Papilio RBP-like proteins in other insects including several species of butterflies, dragonflies, cicadas, grasshoppers and honeybees. Anti-Papilio RBP immunoreactivity was confirmed in the proteins isolated only from butterflies belonging to the superfamily Papilionoidea and not from other species. In all insects tested, however, fluorescing proteins were clearly detected, suggesting that these insects also have similar retinol-binding proteins.