SUMMARY We have previously described the first cloning and partial characterization of carbonic anhydrase from larval Aedes aegypti mosquitoes. Larval mosquitoes utilize an alkaline digestive environment in the lumen of their anterior midgut, and we have also demonstrated a critical link between alkalization of the gut and carbonic anhydrase(s). In this report we further examine the nature of the previously described carbonic anhydrase and test the hypothesis that its pattern of expression is consistent with a role in gut alkalization. Additionally we take advantage of the recently published genome of the mosquito Anopheles gambiae to assess the complexity of the carbonic anhydrase gene family in these insects. We report here that the previously described carbonic anhydrase from Aedes aegypti is similar to mammalian CA IV in that it is a GPI-linked peripheral membrane protein. In situ hybridization analyses identify multiple locations of carbonic anhydrase expression in the larval mosquito. An antibody prepared against a peptide sequence specific to the Aedes aegypti GPI-linked carbonic anhydrase labels plasma membranes of a number of cell types including neuronal cells and muscles. A previously undescribed subset of gut muscles is specifically identified by carbonic anhydrase immunohistochemistry. Bioinformatic analyses using the Ensembl automatic analysis pipeline show that there are at least 14 carbonic anhydrase genes in the Anopheles gambiae genome, including a homologue to the GPI-linked gene product described herein. Therefore, as in mammals which similarly possess numerous carbonic anhydrase genes, insects require a large family of these genes to handle the complex metabolic pathways influenced by carbonic anhydrases and their substrates.