Although a nocturnal rise in PRL secretion is well known, it has long been presumed to be evoked by sleep. To determine whether PRL secretion was driven by a sleep-independent circadian rhythm, we studied 12 men and 10 women using a constant routine protocol. Under the constant routine conditions of continuous semirecumbent wakefulness in constant indoor room light with hourly meals distributed throughout the day and night, a persistent circadian rhythm of PRL secretion was present in men and in women at the follicular and luteal phases of the menstrual cycle. Furthermore, the amplitude of this rhythm in women was significantly greater than that in men. The present data demonstrate the presence of a robust sleep-independent endogenous circadian rhythm of PRL secretion in humans. We hypothesize that this endogenous component of the circadian rhythm of PRL secretion together with those of body temperature, urine production, and cortisol, TSH, and melatonin secretion are driven by the central circadian pacemaker located in the suprachiasmatic nucleus of the hypothalamus.