Immunocytochemical studies on the nervous system,Hatschek’s pit,digestive tract and gonads tissues of an amphioxus(Branchiostoma belcheri)were performed using polyclonal antibodies against human corticotrophin-releasing hormone(CRH)and human adrenocorticotropin(ACTH).The results showed that many CRH-like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicle,while a few CRH-like neurons and their fibers in spinal cord.At the same time,the epithelial cells in the basic region of Hatschek’s pit were shown immunopositive to CRH antibody.In gonads(ovary and testis),CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells.ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord,and also in epithelial cells of Hatschek’s pit,enteric neurons of digestive tract,oocytes in ovary and in early spermatogenic cells as well.It was found for the first time that CRH-like neurons existed in the middle region of brain vesicle(corresponding to the hypothalamus of vertebrates)and ACTH-like immunopositive cells existed in Hatschek’s pit,implying that a control mechanism between brain vesicle and Hatschek’s pit maybe had been already built in amphioxus as that in vertebrates.The present study will provide new morphological evidence for the origin and evolution of ACTH.In addition,the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus. Immunocytochemical studies on the nervous system, Hatschek’s pit, digestive tract and gonads tissues of an amphioxus (Branchiostoma belcheri) were performed using polyclonal antibodies against human corticotrophin-releasing hormone (CRH) and human adrenocorticotropin (ACTH). The results showed that many CRH- like immunoreactive neurons were distributed specifically on the dorsal side and ventral side of brain vesicles, while a few CRH-like neurons and their fibers in spinal cord. At the same time, the epithelial cells in the basic region of Hatschek’s pit were shown immunopositive to CRH antibody. In gonads (ovary and testis), CRH-immunopositive substance was localized in the cytoplasm near oocyte nucleus and in early spermatogenic cells. ACTH-like immunoreactivities were observed specially in the neurons and their protrusions localized on the ventral side of the brain vesicle and in spinal cord, and also in epithelial cells of Hatschek’s pit, enteric neurons of digestive tract, oocytes in ovary and in ea rly spermatogenic cells as well. It was found for the first time that CRH-like neurons existed in the middle region of brain vesicles (corresponding to the hypothalamus of vertebrates) and ACTH-like immunopositive cells existed in Hatschek’s pit, implying that a control mechanism between brain vesicle and Hatschek’s pit maybe had been already built in amphioxus as that in vertebrates. The present study will provide new morphological evidence for the origin and evolution of ACTH. addition, the immunoreactivities of CRH and ACTH in the digestive tract and gonads suggested other physiological function of CRH and ACTH in amphioxus.