Spinosad is a relatively new insecticide that exerts its toxic action via nicotinic acetylcholine receptors (nAChRs). Spinosad resistance in house flies appears to be due to an altered target site. To evaluate the molecular basis of spinosad resistance, two nAChR subunit genes, Mdalpha5 and Mdbeta3 were cloned and characterized from an insecticide-susceptible (aabys) and spinosad resistant (rspin) strain of the house fly, Musca domestica. The Mdalpha5 and Mdbeta3 cDNAs encode proteins of 781 and 432 amino acid residues, respectively. Phylogenetic analysis with insect nAChR subunits suggested that Mdalpha5 and Mdbeta3 are most closely related to Dalpha5 and Dbeta3 of Drosophila melanogaster, respectively. Mdbeta3 is intronless, which is unique among all previously described nAChR genes. A-to-I RNA editing was found at 13 sites in Mdalpha5, eleven of which resulted in amino acid substitutions. No evidence for A-to-I RNA editing was found in Mdbeta3. Mdalpha5 expression, quantified by real time PCR, was 340- and 23-fold higher in the head and thorax than in the abdomen. Mdbeta3 expression was more uniform, being only 2.4-fold higher in the head and 1.4-fold lower in the thorax, compared to the abdomen. There was no difference in the expression of Mdalpha5 and Mdbeta3 between the aabys and rspin strains. Although Mdalpha5 and Mdbeta3 both map to the same chromosome as spinosad resistance, there were no unique features of either gene in rspin, relative to the aabys strain. This suggests neither Mdalpha5 nor Mdbeta3 is responsible for spinosad resistance in house flies.