In the present study the effect of methadone (M) on gonadotropin-releasing hormone in the rat hypothalamus was investigated. The specificity of the M effect was demonstrated by injecting M (100 ng) and the M antagonist, naloxone (5 ng), into the ventromedial hypothalamus. A significant drop in the blood testosterone level (p < 0.05) 15 min following M injection and reversal following naloxone administration suggest a decrease in synthesis or release of LHRH. To determine whether the LHRH response to dopamine (DA) is altered by M, studies were conducted in vitro and in vivo. For the in vitro study hypothalami were incubated with M (10–7 M), DA (10–7 M), DA plus M, and DA plus M plus naloxone. Supernatants from the incubate were used to coincubate pituitaries in order to study the effect on luteinizing hormone release. Treatment of the hypothalamus with M had no effect on basal but inhibited the DA-stimulated release of LHRH. For the in vivo study rats were pretreated with decarboxylase inhibitor (RO4–4602) followed by treatment per kilogram body weight with M (10 mg), L-dopa (1, 10, and 100 mg), L-dopa plus M, LHRH (500 ng), and M plus LHRH. M had no effect on the LHRH-induced increase in testosterone levels, whereas the L-dopa-induced increase in testosterone was blunted by M treatment. It is concluded that M blocks the DA-mediated release of gonadotropin-releasing hormone in the rat hypotha