Galanin and vasopressin are coexpressed in the bed nucleus of the stria terminalis and medial amygdala of the male rat. In adult males, the level of gene expression for both peptides in these regions is dependent on circulating levels of testosterone. We hypothesized that galanin messenger RNA levels would be enhanced in adult males compared with prepubertal males due to the rise in plasma testosterone levels. We used in situ hybridization and quantitative autoradiography to measure galanin messenger RNA in cells of the bed nucleus of the stria terminlis and medial amygdala of prepubertal and adult male rats. Our results show that significantly (P less than or equal to 0.05) more galanin messenger RNA expressing neurons are detectable in the bed nucleus of the stria terminalis of adult compared with prepubertal male rats. In contrast, no differences were observed between the groups in the number of labeled neurons detected within the medial amygdala. However, the average labeling intensity was significantly enhanced in both the bed nucleus of the stria terminalis (P less than or equal to 0.001) and medial amygdala (P less than or equal to 0.001) of adult compared with prepubertal animals. The present findings are consistent with the hypothesis that gonadal hormones regulate galanin gene expression in some brain regions and suggest that the activation of the hypothalamic-pituitary-gonadal axis which occurs naturally with puberty is associated with activation of galanin pathways in the bed nucleus of the stria terminalis and medial amygdala.