The adipokinetic hormones (AKHs) from the corpora cardiaca (CC) of representative species from all three subfamilies of the Sphingidae (hawkmoths) were investigated using matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) and liquid chromatography electrospray ion trap mass spectrometry (LC–ESI MS), including a re-examination of the AKH complement of the tobacco hawkmoth, Manduca sexta. In addition to larvae and adults of M. sexta (subfamily: Sphinginae), adults from the following subfamilies were examined: Macroglossinae (large elephant hawkmoth, Deilephila elpenor), Smerinthinae (poplar hawkmoth, Laothoe populi and eyed hawkmoth, Smerinthus ocellata), and Sphinginae (death′s head hawkmoth, Acherontia atropos). All moths are shown to have the nonapeptide Manse-AKH (pELTFTSSGWamide) in their CC, together with a second AKH, which, on the basis of mass ions ([M+Na]+, [M+K]+) and partial sequence analysis is identical in all species examined. The structure of this AKH was elucidated from peptides leached out of the CC of adult M. sexta and shown, by ESI-collision-induced dissociation (CID) tandem mass spectrometry (MS/MS), to be a novel decapeptide AKH with a sequence of pELTFSSGWGQamide. The new peptide has been code named Manse-AKH-II. Sequence confirmation was obtained from identical MS studies with synthetic Manse-AKH-II and with the native peptide. Manse-AKH-II has significant lipid-mobilizing activity when injected at low dose (5 pmol) into newly emerged adult M. sexta. The potential implications of a second AKH, in M. sexta in particular, are discussed in relation to putative receptor(s).