Since there are several endogenous morphine-like substances differing in distribution and probably also in function, it seems necessary to study their roles in acupuncture analgesia separately. Therefore we set up a sensitive and specific radioimmunoassay (RIA) for met- and leu-enkephalin, and employ it in determining the changes of enkephalin contents in the brain regions and CSF during acupuncture. The main results are as follows: (i) Two enkephalins increase markedly in hypothalamus and striatum during acupuncture analgesia. (ii) Prior intraventricular bacitracin enhances the acupuncture analgesia with concomitant increased contents of enkephalins in the brain and CSF. (iii) Cycloheximide, the protein-synthesis inhibitor, reduces the enkephalin-increasing effect of acupuncture, indicating that one of the mechanisms by which acupuncture elevates the enkephalin levels is the acceleration of biosynthesis. (iv) After surgical isolation of rat hypothalamus, the effect of electric acupuncture is attenuated. This indicates that hypothalamus takes part in acupuncture analgesia.