Mosquito larvae generate a luminal pH as high as 10.5 in the anterior region of their midgut. The mechanisms responsible for the generation and maintenance of this alkaline pH are largely unknown, but there is evidence suggesting a role for the enzyme carbonic anhydrase (CA). CA has been cloned from the alimentary canal epithelium of Anopheles gambiae larvae and can generate bicarbonate, which is implicated as a buffer for the larval lumen. The question remains as to how the bicarbonate is transported from the cells into the lumen. We hypothesize the presence of a CA within the lumen itself to generate bicarbonate from CO(2) produced by the metabolically active alimentary canal cells. Here, we report the cloning and characterization of a novel cytoplasmic-type alpha-CA from the larval An. gambiae alimentary canal. Antibody immunolocalization reveals a unique protein distribution pattern that includes the ectoperitrophic fluid, 'transitional region' of the alimentary canal, Malpighian tubules and a subset of cells in the dorsal anterior region of the rectum. Localization of this CA within the lumen of the alimentary canal may be a key to larval pH regulation, while detection within the rectum reveals a novel subset of cells in An. gambiae not described to date. Phylogenetic analysis of members of the alpha-CA family from the Homo sapiens, Drosophila melanogaster, Aedes aegypti and An. gambiae genomes shows a clustering of the novel CA with Homo sapiens CAs but not with other insect CAs. Finally, a universal system for naming newly cloned An. gambiae CAs is suggested.