An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I--the neuronal alpha-bungarotoxin binding-site subunits (alpha 7, alpha 8); III--the neuronal nicotinic subunits (alpha 2-alpha 6, beta 2-beta 4), which also contain the muscle acetylcholine-binding subunit (alpha 1); and IV--the muscle non-alpha subunits (beta 1, gamma, delta, epsilon). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.