Abstract: The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicaeα-subunit cDNAs, Mpα1 and Mpα2, have been cloned previously. Here we report the isolation of three novel α-subunit genes (Mpα3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect α subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-α subunits are clearly different from vertebrate neuronal β and muscle non-α subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR α-subunit cDNAs with the rat β2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the α subtype. This is the first demonstration that imidacloprid selectively acts on Mpα2 and Mpα3 subunits, but not Mpα1, in M. persicae.