Throughout a 24-h period, immunoreactive melatonin concentrations in Harderian glands of female golden hamsters were approximately 200 pg/mg protein with a significant decline to 80 pg/mg protein only at about 0600, 2 h after light on. Concentrations in glands of males are diurnally constant and low (ca. 20 pg/mg protein). Castration increases immunoreactive melatonin in glands of males to female levels. While blinding alone had no effect, it did prevent the castration-induced increase. Lower concentrations were measured in glands of blinded or blinded ovariectomized females but this decrease was not significant. These data suggest that immunoreactive melatonin concentrations in the Harderian glands of hamsters are controlled by testosterone or its derivatives; these same factors also control the male or female character of these glands.