Endothelin-1 (ET-1) is known as a vasoconstrictor. Several studies have reported that ET-1 induced pain signals through the ETA receptor (ETAR). Although ETAR antagonists are reported to enhance analgesic effects of opioid agonists and relieve its tolerance, the mechanisms remain unclear. We previously revealed the novel and highly selective ETAR antagonist (provided by Eisai Co., Ltd.) enhanced morphine-induced opioid receptor (OR) activities with HEK293 cells stably expressing either ETAR (ETAR cells), μOR (μOR cells), or both ETAR and μOR (ETAR/μOR cells). Further, we found that μOR and ETAR form a dimer as a possible target of this antagonist, and ET-1 internalized the ETAR/μOR heterodimer into the cytosol, resulting in decreased numbers of μOR on the cell membrane, followed by reduced responses by morphine. There are opioid agonists prone to internalize μOR or not; morphine is hardly to internalize, whreas fentanyl preferentially internalizes μOR among opioid agonists. Such difference in μOR internalization is believed to involve in the analgesic tolerance. In the present study with cells expressing ETAR/μOR, we evaluated the effects of the ETAR antagonist on the enhancement of μOR activities by several opioid agonists in addition to morphine, and compared the selectivity of the ETAR antagonist as an analgesic adjuvant.