Localization of melatonin in the pineal glands of 1, 21, 56 and 560dayold chickens kept under 12hr light and 12hr dark photoperiodic conditions was examined. Pineal parenchymal cells of the chicken were immunostained using a primary antimelatonin antiserum at dilutions up to 1:5, 000. The antiserum recognized melatonin specifically in situ, although crossreactivity with related compounds particularly with 5-methoxytryptamine hydrochloride was observed in vitro. Immunoperoxidase method for the demonstration of melatonin was applied to paraffin sections of icecold 10% neutral buffered formalin or BOUIN's fixation in the chicken pineal gland. A more intense immunostaining was found at 12:00 (midday) in the follicular zone as compared with the parafollicular zone in the pineal gland of al dayold chick, but no difference between these two divided zones was observed in immunoreactivity after 21 days of age. In both follicular and parafollicular cells at 24:00 (midnight), intense immunostaining in almost all the parenchymal cells was observed at all ages examined in our study. The number of immunoreactive melatonin granules in these two regions of pineal tissue thus showed daily changes. The amount of immunoreactive melatonin in the pineal parenchyma increased both at midday and midnight with advancing age. These results suggested that the regression of the photosensory function paralleled by crushing follicular structure resulted in the progression of the competence of melatonin biosynthesis with advancing age.