Effects of intraventricular injection of endomorphin-1, endomorphin-2 and β-endorphin on the release of immunoreactive [Met 5 ]enkephalin from the spinal cord were studied in pentobarbital anesthetized rats. Intraventricular injection of endomorphin-2, but not endomorphin-1, caused an increased release of immunoreactive [Met 5 ]enkephalin in the spinal perfusates. β-Endorphin given intraventricularly also increased the release of immunoreactive [Met 5 ]enkephalin in an amount 15-fold higher than that produced by endomorphin-2. The increase of the release of immunoreactive [Met 5 ]enkephalin induced by endomorphin-2 was blocked by μ-opioid receptor antagonist CTOP. Our result suggests that endomorphin-2 stimulates another subtype of μ-opioid receptor different from that acted by endomorphin-1 at the supraspinal site and subsequently increases the release of [Met 5 ]enkephalin from the spinal cord.