The horn fly, Haematobia irritans L., is an obligate blood-feeding parasite of cattle, and control of this pest is a continuing problem because the fly is becoming resistant to pesticides. Dominant conditional lethal gene systems are being studied as population control technologies against agricultural pests. One of the components of these systems is a female-specific gene promoter that drives expression of a lethality-inducing gene. To identify candidate genes to supply this promoter, microarrays were designed from a horn fly expressed sequence tag (EST) database and probed to identify female-specific and larval-specific gene expression. Analysis of dye swap experiments found 432 and 417 transcripts whose expression levels were higher or lower in adult female flies, respectively, compared with adult male flies. Additionally, 419 and 871 transcripts were identified whose expression levels were higher or lower in first-instar larvae compared with adult flies, respectively. Three transcripts were expressed more highly in adult females flies compared with adult males and also higher in the first-instar larval lifestage compared with adult flies. One of these transcripts, a putative nanos ortholog, has a high female-to-male expression ratio, a moderate expression level in first-instar larvae, and has been well characterized in Drosophila. melanogaster (Meigen). In conclusion, we used microarray technology, verified by reverse transcriptase-polymerase chain reaction and massively parallel pyrosequencing, to study life stage- and sex-specific gene expression in the horn fly and identified three gene candidates for detailed evaluation as agene promoter source for the development of a female-specific conditional lethality system.