Regional responses of rat brain opioid receptors (Op-Rs) upon castration and testosterone replacement were studied using [H-3]naloxone (NAL) binding for u-type and [H-3]D-Ala2-methionine-enkephalin (ENK) binding for delta-type Op-R. Castration itself produced an increase in specific NAL and ENK binding in the olfactory bulb and thalamus+midbrain. In the hypothalamus, specific ENK binding was increased without any change in NAL binding. After the testosterone replacement in castrated rats, specific NAL binding was decreased and returned to the control level in the thalamus+midbrain, but remained high in the olfactory bulb. Specific ENK binding was decreased and returned to the control level in the olfactory bulb, hypothalamus and thalamus+midbrain. These results indicated that there are regional differences between u- and delta-type Op-R responses to castration and testosterone replacement. Op-R subtypes were regulated differentially by endogenous hormonal changes, such as testosterone.