Lysozymes have been described in invertebrates as digestive or immune molecules. We report here the characterization of two novel c-type lysozymes, RpLys-A (EU250274) and RpLys-B (EU250275), isolated from the fat body and digestive tract of immune stimulated Rhodnius prolixus, a major vector of Chagas disease. Transcriptional profiles indicate that the temporal and spatial expression patterns of these two peptides are very different. RpLys-A is expressed predominantly in the midgut after ingestion of Trypanosoma cruzi in a bloodmeal, or after injection of bacteria into the hemocoel. RpLys-B is expressed primarily in the fat body after bacterial injection. Phylogenetic alignments indicate that RpLys-A aligns best with molecules from other hemipterans whose major expression is found in the intestinal tract whereas RpLys-B aligns best with mosquito and tick molecules whose expression is found principally in hemocytes and fat body and whose role has been described as immune-related. These data suggest a differential compartmentalized role of two closely related molecules; one for immunity in the hemocoel and the other for digestion in the midgut.