The photoneuroendocrine system translates environmental light conditions into the circadian production of endocrine and neuroendocrine signals. Central to this process is the pineal organ, which has a conserved role in the cyclical synthesis and release of melatonin to influence sleep patterns and seasonal reproduction. In lower vertebrates, the pineal organ contains photoreceptors whose activity entrains an endogenous circadian clock and regulates transcription in pinealocytes. In mammals, pineal function is influenced by retinal photoreceptors that project to the suprachiasmatic nucleus-the site of the endogenous circadian clock. A multisynaptic pathway then relays information about circadian rhythmicity and photoperiod to the pineal organ. The gene cone rod homeobox (crx), a member of the orthodenticle homeobox (otx) family, is thought to regulate pineal circadian activity. In the mouse, targeted inactivation of Crx causes a reduction in pineal gene expression and attenuated entrainment to light/dark cycles. Here we show that crx and otx5 orthologs are expressed in both the pineal organ and the asymmetrically positioned parapineal of larval zebrafish. Circadian gene expression is unaffected by a reduction in Crx expression but is inhibited specifically by depletion of Otx5. Our results indicate that Otx5 rather than Crx regulates genes that show circadian expression in the zebrafish pineal complex.