This study analyzes the effect of chronic ethanol feeding on 24-h variation of hypothalamic-pituitary mechanisms involved in prolactin regulation in growing male Wistar rats. Animals were maintained under a 12:12 h light/dark photoperiod (lights off at 2000 h), and they received a liquid diet for 4 wk, starting on d 35 of life. The ethanol-fed group received a similar diet to controls except that maltose was isocalorically replaced by ethanol. Ethanol replacement provided 36% of the total caloric content of the diet. Rats were killed at six time intervals every 4 h, beginning at 0900 h. Mean concentration of serum prolactin in ethanol-fed rats was 58.7% higher than in controls. Peak circulating prolactin levels occurred at the early phase of the activity span in both groups of rats, whereas a second peak was found late in the resting phase in ethanol-fed rats only. In control rats, median eminence dopamine (DA), serotonin (5-HT), gamma-aminobutyric acid (GABA), and taurine levels exhibited two maxima, the major one preceding prolactin release and a second one during the first part of the resting phase. Median eminence DA and 5-HT turnover (as measured by 3,4-dihydroxyphenylacetic acid, DOPAC/DA, and 5-hydroxyindoleacetic acid, 5-HIAA/5-HT ratio) showed a single maximum preceding prolactin, at 0100 h. Ethanol treatment did not affect median eminence DA or 5-HT levels but it decreased significantly their turnover rate. The midday peak in DA and 5-HT levels (at 1300 h) was abolished and the night peak (at 0100 h) became spread and blunted in the ethanol-fed rats. This was accompanied with the disappearance of the 0100 h peak in DA and 5-HT turnover and the occurrence of a peak in 5-HT turnover at 1700 h. Ethanol intake suppressed the night peak in median eminence GABA and taurine (at 0100 h) as well as the midday peak of GABA. Ethanol augmented pituitary levels of DOPAC and 5-HIAA. The results indicate that chronic ethanol administration affects the mechanisms that modulate the circadian variation of prolactin release in growing male rats.