Abstract Rhodnius prolixus undergoes a period of rapid diuresis after ingesting large blood meals. Neurohormones with either diuretic or anti-diuretic activity control diuresis by acting on several tissues including the Malpighian tubules. One of the neurohormones that potentially plays a role in diuresis is diuretic hormone 31 (DH 31) which belongs to the insect calcitonin-like family of diuretic hormones. Here we determine the complete cDNA sequences of three Rhopr-DH 31 splice variants ( Rhopr-DH 31 -A, Rhopr-DH 31 -B and Rhopr-DH 31 -C) and characterize their expression in unfed fifth-instar R. prolixus. Reverse transcriptase-PCR demonstrates that Rhopr-DH 31 is predominantly expressed in the central nervous system (CNS) of unfed fifth-instars. However, the expression of the three splice variants differs with Rhopr-DH 31 -B expression being the highest followed by Rhopr-DH 31 -A and Rhopr-DH 31 -C, as determined using semi-quantitative Southern blot analysis. Fluorescent in situ hybridization reveals that Rhopr-DH 31 is expressed in a variety of cells in the CNS, including some neurosecretory cells.