The characteristics of kyotorphin (Tyr-Arg)-induced release of Met-enkephalin from the striatum and the spinal cord of guinea pig were determined by superfusing the slices in vitro and then carrying out radioimmunoassays. Depolarization by 50 mM K+ induced a marked release of Met-enkephalin-like immunoreactivity. The potassium-induced release of Met-enkephalin was calcium-dependent. In preparations from a striatum, the addition of kyotorphin to the superfusion medium produced a concentration-dependent increase in Met-enkephalin. The kyotorphin-induced release of Met-enkephalin was calcium-dependent and was abolished by tetrodotoxin. Similar effects of kyotorphin were seen in the spinal cord preparations. Electrical field stimulation of the striatal slices at a frequency of 10 Hz also evoked significant and calcium-dependent increases in the release of Met-enkephalin and markedly enhanced the kyotorphin-induced release of Met-enkephalin, as compared to the controls not given field stimulation. These results suggest that kyotorphin depolarizes the so-called enkephalinergic neurons and releases Met-enkephalin from nerve terminals. This effect of kyotorphin may be a possible mechanism related to the manifestation of analgesia.