Insect prothoracicotropic hormone (PTTH) is a cerebral neurohormone which is synthesized in the specific neurosecretory cells. In present study, the PTTH was partially purified from D. melanogaster and its regulatory roles on activation of target tissues were investigated by the in vitro culture of prothoracic glands and by the radioimmunoassay. Also, using BSA or RSA as ecdysteroid-carrier proteins, we prepared two kinds of anti-ecdysteroid rabbit antisera. As the results, the molecular weight of PTTH was less than 22 kD, and treatment of PTTH on culture medium of prothoracic glands increased the production of ecdysteroid. In following experiment, addition of ecdysteroid to in vitro culturing tissues such as imaginal discs and salivary glands induced the synthesis of Hsp23. Consequently, we could observe the brain (PTTH) - prothoracic gland (ecdysteroid) - target (Hsp23) endocrine axis previously known. But, we showed that the PTTH also directly stimulated the imaginal wing disc to produce Hsp83. This result suggested that another brain (PTTH) - target (Hsp83) axis distinguished from the upper axis was existed.