The analgesic effect of morphine sulfate (3-5 mg/kg, i.p.) was assessed by both tail-flick and hot-plate tests in unanesthetized restrained rats. Intrathecal administration of atropine sulfate (10 micrograms) in the lumbar region of the spinal cord powerfully reduced the analgesia induced by systemic administration of morphine. This action did not result from the diffusion of atropine from its administration site to more rostral sites in the central nervous system. In spinal rats, atropine failed to reverse morphine analgesia, thus strongly suggesting that either a cholinergic descending pathway or a spinal local cholinergic circuit activated by an unknown descending pathway may be involved in the systemic morphine analgesia. In addition, the involvement of the alpha-adrenergic descending pathway in morphine analgesia is confirmed, whereas that of the serotonergic descending pathway is less prominent.