Ion transport peptide (ITP) in insect known for the role in water reabsorption and ecdysis in Arthropod. We identified conserved itp genes containing alternative splicings in Drosophila melanogaster and Anopheles gambiae genome sequences. The transcripts from three alternative splicings putatively produce two different C-terminally acid forms (DmITPL1, and DmITPL2) and an amidated isoform (DmITP) in D. melanogaster, while only two alternatively spliced forms encoding each one of C-terminally acid and amidated forms (AgITPL and AgITP, respectively) were identified in A. gambiae. Immunohistochemistry and in situ hybridization found the ITP commonly expressed in the four pairs of brain cells having projection to corpora cordiaca and ITPL in the peripheral nervous system. A putative D. melanogestar itp mutant EP (2)2287 is deficient in trachea airfill at the late embryonic stage, implying deficiency in removal of fluid in trachea tube. Mutant analysis of the EP (2)2287 showed an obvious deficiency in the expression of DmITP (C-terminal amidated form) among three different isoforms. Overexpression of the DmITP and DmITPL by using GAL4 drivers induced lethality at the early larval stages. Current data suggest that the function of ITP includes anti-diuresis and ITPL has a novel function unknown yet.