Repeated administration of morphine or orexin-A produces tolerance to their antinociceptive effects. We investigated the possible incidence of cross-tolerance between orexin-A and morphine. Adult male Sprague-Dawley rats (200–250 g) were used. Under deep anesthesia, a stereotaxic apparatus was used to implant a 23 G cannula into the lateral ventricle for an intracerebroventricular (ICV) microinjection. The antinociceptive effect of three different doses of orexin-A (5, 20, and 40 µM; dissolved in 5 µl sterile saline; ICV) was examined using the hot-plate test at 15, 30, 60, and 90 min after infusion. To evaluate tolerance, orexin-A (20 µM; ICV) or morphine (10 mg/kg; intraperitoneal) was administered for 7 consecutive days (twice per day) and the analgesic response was assessed at days 1, 4, and 7. Cross-tolerance was investigated at day 8 with a single injection of morphine (10 mg/kg; intraperitoneal) to the repeated orexin-A group and a single microinjection of orexin-A (20 µM; ICV) to the repeated morphine group. Analgesic responses were then examined. Administration of both orexin-A and morphine produced significant antinociception at day 1 (P<0.001 compared with the saline group). However, a significant reduction in the analgesic effects of both morphine and orexin-A appeared at day 7, following repeated administration (P<0.01). Orexin-A microinjection at day 8 in the repeated morphine group did not result in significant antinociception (P>0.05), whereas morphine injection in the repeated orexin-A group at day 8 showed a significant analgesic effect (P<0.001). These results indicate cross-tolerance to the analgesic effect of orexin-A following morphine tolerance.