Diuresis in the blood-gorging hemipteran Rhodnius prolixus is under neurohormonal control and involves a variety of processes and tissues. These include ion and water movement across the epithelium of the crop and the Malpighian tubules, and muscle contractions of the crop, hindgut and dorsal vessel, which facilitate mixing of the blood-meal, mixing of the haemolymph, as well as the expulsion of waste. One of the neurohormones that might play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH(31)) family of insect peptides. Previously we have demonstrated the presence of DH(31)-like peptides in the central nervous system (CNS) and gut of R. prolixus 5th instars. In the present work, a DH(31) from the CNS of 5th instar R. prolixus was isolated using reversed-phase liquid chromatography (RPLC), monitored with an enzyme-linked immunosorbent assay (ELISA) combined with matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry, and sequenced using tandem mass spectrometry and Edman degradation. This neuropeptide is the first to be sequenced in R. prolixus and has a sequence identical to that found previously for Dippu-DH(31) from the cockroach Diploptera punctata. In previous studies testing Rhopr/Dippu-DH(31) in Malpighian tubule secretion assays, we demonstrated increases in the rate of secretion that were small, relative to that induced by serotonin, but nevertheless 14-fold over baseline. In the present study, we investigated second messenger pathways in response to Rhopr/Dippu-DH(31) and found no increase or decrease in cyclic adenosine monophosphate (cyclic AMP) content of the Malpighian tubules. DH(31)-like immunoreactivity is present over the dorsal hindgut, anterior dorsal vessel and dorsal diaphragm, and bioassays of the R. prolixus dorsal vessel and hindgut indicate that Rhopr/Dippu-DH(31) increases the frequency of muscle contractions of both tissues. Second messenger pathways were also investigated for the dorsal vessel and hindgut.