Insulin-like peptides are key regulators of metabolism, reproduction, and senescence in higher eukaryotic organisms. Here we present the identification, expression, and tissue localization of eight genes encoding insulin-like peptides (ILPs) in the yellow fever mosquito, Aedes aegypti. All eight ILPs share the conserved features of the insulin superfamily as prepropeptides consisting of contiguous signal, B, C, and A peptides. However, one of the ILPs has a truncated C peptide and a carboxy terminal extension, features consistent with insulin growth factors. Transcripts for five of the ILPs occurred predominantly in the heads (brains) of larval, pupal, and adult mosquitoes. Transcripts of two other genes, one of which was the putative insulin growth factor, were present in the head, thorax and abdomens of all stages. The final ILP was predominantly expressed in abdomen. Results from immunocytochemistry with two different ILP antisera showed cellular localizations in the nervous system and midgut that corroborated the existence of these expression patterns. Three of the ILP genes are so closely linked that only the 5' region of the first ILP gene likely suffices as a promoter, indicating that these genes form a eukaryotic operon. The nearly identical expression pattern of these three ILPs supported this idea. Finally, the phylogenetic relationship of ILPs from three dipteran species, Ae. aegypti, the African malaria mosquito (Anopheles gambiae), and Drosophila melanogaster is presented as a step towards understanding the structural and functional diversity of insect ILPs.