Ethanol has been known to suppress reproductive activity in laboratory animals and humans through the inhibition of luteinizing hormone (LH) release by reduction of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. There are, however, little data is available regarding the effect of ethanol on GnRH gene expression. Thus, the present study was designed to evaluate the effect of ethanol on GnRH gene expression and reproductive activity at all levels of the hypothalamus-pituitary-gonad (HPG) axis simultaneously. To this end, ethanol (3 g/kg i.p., 15% v/v in saline) was administered to adult male rats for 10 days. Serum levels of LH and testosterone were significantly decreased by ethanol. Using Northern blot analysis and in situ hybridization, the present study showed the reduction in GnRH mRNA levels in the hypothalamus by prolonged ethanol administration. The content of LH in the anterior pituitary was also significantly reduced by ethanol. In addition, steroidogenic acute regulatory protein (StAR) mRNA levels were significantly decreased by ethanol, suggesting a cause for the reduced production of testosterone under this condition. These results indicate that ethanol affects the HPG axis at all the levels. Especially, suppressed GnRH mRNA levels in the hypothalamus of ethanol-treated rats strongly demonstrated that hypothalamus is the major action site of ethanol on the HPG axis. Decreased serum LH level may affect the steroidogenesis in the testis, at least in part, through the inhibition of StAR gene expression that induces part of dysfunctions of reproductive activity.