The gene encoding the last enzyme of the melatonin-synthesis pathway, hydroxyindole-O-methyltransferase (HIOMT), is selectively expressed in retinal photoreceptors and pineal cells. Here, we analysed the promoter of the chicken HIOMT gene and we found that a homeodomain-binding element located in the proximal region of this promoter was essential for its activation in primary cultures of embryonic chicken retinal cells. This homeodomain-regulatory element interacted with a protein expressed in the chicken retina and pineal gland, which was recognized by an anti-Otx2 antiserum. Recombinant Otx2 expressed in vitro was able to bind this DNA element and to directly transactivate the chicken HIOMT promoter. This promoter was also transactivated by another member of the Otx family, Otx5, but the amplitude of stimulation was lower than with Otx2. The spatio-temporal pattern of Otx2 expression was compatible with a possible role of this transcription factor in HIOMT gene activation. In adult chicken, Otx2 mRNA was found to be present in those two tissues that express HIOMT: the retina and the pineal gland. During development, a burst of Otx2 mRNA closely matched the timing of HIOMT gene activation in these two tissues. In the pineal, Otx2 immunolabelling was specifically localized in the nuclei of photoreceptor cells. In the neural retina, Otx2 immunoreactivity brightly decorated the photoreceptor nuclei and extended more faintly to the outer half of the inner nuclear layer. Together, the data support a role of Otx2 in the onset of HIOMT expression in developing chicken photoreceptors.