This work analyzes the effect of chronic ethanol feeding on the 24-h variation of mitogenic responses and lymphocyte subset populations in thymus and spleen. Animals were maintained under a 12:12 h light/dark photoperiod and they received a liquid diet for 4 weeks, starting on day 35 of life. The ethanol-fed group received a similar diet to controls except for that maltose was isocalorically replaced by ethanol. Ethanol replacement provided 36% of the total caloric content of the diet. Rats were humanely killed at 6 time intervals around the clock, beginning at Zeitgeber time (ZT) 1 (ZT 0 = lights on). Under ethanol intake, both splenic and thymic weights decreased. Also, mean values of thymic, but not of splenic T cell number decreased, and mean values of thymic and splenic CD8+ and CD4+-CD8+ number augmented. Consequently, thymic T/B ratio and thymic and splenic CD4+/CD8+ ratio decreased in ethanol-fed rats. These findings coexisted with a significant increase in thymic cells response to LPS. The ethanol diet modified 24 h rhythmicity of thymic and splenic T, B and CD4+-CD8+ cells, thymic CD4+ and splenic CD8+ cells, thymic and splenic T/B and CD4+/CD8+ ratios, as well as of mitogenic responses in both tissues. Chronic ethanol administration presumably affects the endogenous clock that modulates the circadian variation of immune responsiveness in growing rats.