The production of ecdysteroid by the insect prothoracic glands (PTGs) is initiated by an ecdysiotropin, prothoracicotropic hormone, which is produced by the brain and released into the hemolymph from its neurohemal organ. Recently we reported the discovery of another ecdysiotropin which is localized in the lepidopteran hindgut or proctodaeum. It is a small, heat-stable peptide which is resistant to freeze-thaw and to extraction with organic solvents. Based on size-exclusion HPLC, we now estimate its molecular weight to be 500-1500 Da. The hindgut ecdysiotropin stimulates the PTGs of Lymantria dispar to produce both ecdysone and 3-dehydroecdysone in a dose-dependent manner. Ecdysteroid production was maximum in the presence of 0.125 and 0.1 hindgut equivalents for Ostrinia nubilalis and L. dispar, respectively. Activity was detected throughout the pylorus and anterior intestine of the O. nubilalis hindgut. When proctodaea from 5th instar O. nubilalis were analyzed daily for ecdysiotropic activity, those from wandering larvae which had undergone gut purge were found to have the greatest concentration of ecdysiotropin. Cyclic AMP appears to act as a second messenger for the proctodaeal ecdysiotropin as evidenced by the increased levels of cAMP present in PTGs incubated with hindgut extract. At doses which caused maximum stimulation, effects of brain and proctodaeal extracts were additive indicating that the two ecdysiotropins utilize separate receptors. Size exclusion HPLC of hemolymph obtained from prepupae that have experienced gut purge revealed the presence of an ecdysiotropin(s) whose molecular weight range is similar to that of the proctodaeal ecdysiotropin but not to that of the small form of brain PTTH. While the physiological function of the proctodaeal ecdysiotropin(s) is unknown, the discovery of such a peptide(s) is noteworthy in light of the reported production of ecdysteroids by isolated insect abdomens.