A daily rhythm in melatonin concentration in Harderian glands (HG) of female golden hamsters is described and is characterized by significant reductions following lights‐on. Concentrations in HG of intact or pinealectomized males are low and consistent over a 24‐hour period. Castration of males is accompanied by increased levels of melatonin in the HG to approach those of females. When castrated males are exposed to short photoperiods, however, melatonin levels are typically low. Levels of serotonin (5‐HT), 5‐hydroxytryptophan (5‐HTP), and 5‐hydroxyindoleacetic acid (5‐HIAA) are extremely variable in HG of both males and females, precluding definitive conclusions. Melatonin concentrations in HG of females do not appear to be affected by light or stress per se, nor are the superior cervical ganglia the pathway by which concentrations