The in vitro release of LHRH from hypothalami of adult male rats (intact, 5-day castrates, 5-day castrates replaced with various doses of testosterone) was measured under basal conditions and after the addition of KC1, the opiate antagonist naloxone or the opiate agonist DAGO to the perifusion medium. Hypothalami from all treatment groups responded to 56 mM KCl with an increased output of LHRH. LHRH release was also induced by naloxone (10–6M), but only from tissues derived from intacts and castrates given physiological doses of testosterone. The opiate agonist DAGO (10–6M) did not alter the basal release of LHRH; it, however, caused a significant decrease in the K+-induced release of LHRH from hypothalami derived from intact rats and rats replaced with physiological levels of testosterone but not from those derived from castrate rats or castrate rats replaced with small amounts of testosterone. The specificity of this latter response was shown by its reversibility with naloxone. The lack of DAGO effects upon tissues from rats with low levels of steroid implied steroid dependency of the response to opioidergic influences and indeed, the response to DAGO was restored when testosterone was replaced at physiological doses. Measurement of hypothalamic LHRH content showed no significant differences between tissues obtained from intact, castrate and testosterone-replaced castrate rats. These in vitro data support the view that the inhibitory influence of opioids upon LHRH release depends on the presence of gonadal steroids in v