Neuropeptides related to mammalian neuropeptide Y (NPY) and insect neuropeptide F (NPF) are conserved throughout Metazoa and intimately involved in a wide range of biological processes. In insects NPF is involved in regulating feeding, learning, stress and reproductive behavior. Here we identified and characterized an NPF receptor of the tsetse fly, Glossina morsitans morsitans, the sole transmitter of Trypanosoma parasites causing sleeping sickness. We isolated cDNA sequences encoding tsetse NPF (Glomo-NPF) and its receptor (Glomo-NPFR), and examined their spatial and temporal expression patterns using quantitative PCR. In tsetse flies, npfr transcripts are expressed throughout development and most abundantly in the central nervous system, whereas low expression is found in the flight muscles and posterior midgut. Expression of npf, by contrast, shows low transcript levels during development but is strongly expressed in the posterior midgut and brain of adult flies. Expression of Glomo-npf and its receptor in the brain and digestive system suggests that NPF may have conserved neuromodulatory or hormonal functions in tsetse flies, such as in the regulation of feeding behavior. Cell-based activity studies of the Glomo-NPFR showed that Glomo-NPF activates the receptor up to nanomolar concentrations. The molecular data of Glomo-NPF and Glomo-NPFR paves the way for further investigation of its functions in tsetse flies.