Chronic exposure of pubertal male rats to ethanol results in a decline in serum testosterone, increased gonadotropins, pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) content, and decreased or inappropriately normal serum LH and FSH levels, suggesting impaired secretory release of gonadotropins. The molecular mechanisms behind this disorder are undefined, but a disruption of vesicle-mediated secretory processes is possible because intracellular protein trafficking pathways are involved in secretion of glycoproteins such as FSH and LH. Because small GTP-binding proteins of Rab family have been implicated as key regulators of membrane and protein trafficking in mammalian cells, this study was designed to test if ethanol-impaired pituitary FSH and LH secretion is associated with changes in Rab proteins, particularly Rab1B, Rab3B, Rab6, and Rab11. Male Sprague-Dawley rats 35 days old were pair-fed a Lieber-DeCarli diet with ethanol or without ethanol for 5 to 60 days. After ethanol exposure, serum testosterone levels decreased while LH and FSH were inappropriately unchanged. Immunohistochemical staining showed decreased Rab1B, Rab3B, and Rab11 protein levels in ethanol-treated pituitaries. Immunoblotting showed that ethanol induced a transient reduction in Rab6 after 5 days of ethanol exposure, whereas Rab3B decreased after 20 days, Rab11 after 30 days, and Rab1B after 60 days. Despite these changes in Rab proteins, mRNA levels were unaffected by ethanol exposure. We concluded that reductions in key Rab proteins may lead to altered vesicle trafficking and may play a role in disruption of pituitary FSH and LH secretion caused by ethanol.