In many species living in temperate zones, reproduction is controlled by the photoperiod. Recent findings have clarified that type 2 iodothyronine deiodinase (Dio2) plays a significant role in the photoperiodic response of gonads in the mediobasal hypothalamus, converting the prohormone T₄ into bioactive T₃. In mammals, the Dio2 expression is suppressed by long-term melatonin injections, although the signal transduction pathways that link the melatonin signal to the Dio2 expression are unknown. As a first step to approach the problem, we have here investigated the temporal dynamics of the melatonin effect on Dio2 expression using male Syrian hamsters. Dio2 mRNA levels were found to show diurnal rhythms under long-day condition in an area adjacent to the tuberoinfundibular sulcus (TIS) and in the ependymal cell layer lining the ventrobasal walls of the third ventricle (EC). Daily sc melatonin injections given in the late afternoon under long-day condition suppressed the Dio2 mRNA levels already at the first day after the onset of the treatment in the EC, and one day later in the TIS. These suppressive effects were sustained for at least two days after a single injection. Further, we examined the temporal changes of the Dio2 expression after the onset of the treatment, showing that the suppression did not occur until mid-day of the next day. These data suggest that melatonin is involved in the signal transduction mechanisms controlling the photoperiodic response of gonads by acting on the Dio2 expression rather rapidly through indirect pathways.