Ayu (Plecoglossus altivelis) is a teleost whose gonadal development is stimulated by shortened daylength and is a useful model to study the mechanism of photoperiodism. However, localization and characteristics of the photoreceptor that mediates photoperiodism in gonadal development remain to be determined. To identify the photoreceptive molecule that regulates photoperiodic responses, in the present study, we have cloned and characterized the cDNA encoding an opsin gene expressed in the ayu brain, a putative site of the photoreceptor for photoperiodism. The identified opsin was rhodopsin that is identical to the rhodopsin expressed in the retina. Phylogenetic analysis demonstrated that this rhodopsin belongs to the retina-type but not to the pineal-specific rhodopsin group. Genomic polymerase chain reaction (PCR) demonstrated that the ayu rhodopsin gene is intron-less. Southern and Northern blots and reverse-transcription PCR analyses indicate that the same rhodopsin gene is expressed in the retina and the brain but not in the pineal organ of ayu. These results indicate that the rhodospin gene is expressed in the retina and brain and mediates not only visual but also nonvisual functions such as photoperiodism and entrainment of the circadian clock.