The pineal hormone melatonin has been implicated in the regulation of circadian rhythms and in photoperiodic control of reproduction. The effects of melatonin require the hypothalamic suprachiasmatic nucleus (SCN), a principal pacemaker controlling circadian rhythms. To determine whether SCN activity was directly affected by exogenous melatonin, rats received either melatonin or saline injections 15 min before administration of 2-deoxy-[1-14C]glucose (2-DG) at two times of day, circadian time (CT) 10 and CT14, and the brains of these rats were processed for autoradiographic determination of 2-DG uptake within the SCN. We report that SCN 2-DG uptake was inhibited by melatonin at CT10, when 2-DG uptake is normally high, and unaffected at CT14, when 2-DG uptake is normally low. This indicates that the SCN may be neural substrates through which melatonin exerts at least some of its effects on mammalian physiology.