We have isolated and characterized two diuretic hormones (DH), Hylli-DH41 and Hylli-DH30, from extracts of whole heads of the lepidopteran Hyles lineata. We monitored the isolation by measuring the ability of fractions to affect levels of cyclic AMP production by Malpighian tubules of Manduca sexta maintained in vitro. These DH are related to a family of vertebrate neuropeptides which includes sauvagine, corticotropin-releasing factor (CRF), and urotensin I. Both Hylli-DH41 (RMPSLSIDLPMSVLRQKLSLE KERKVQALRAAANRNFLNDI-NH2) and Hylli-DH30 (SFSVNPAVEILQHRYMEKVAQNNRNFLNRV-NH2) show extremely high similarity with two DH from the tobacco hornworm M. sexta. This is not surprising because both H. lineata and M. sexta are sphingid moths. The discovery of these DH provides a third example of two CRF-related DH occurring in one insect species.