We present herein the cloning of the human nicotinic acetylcholine receptor alpha9-ortholog and the identification of a new alpha-like subunit (alpha10) that shares 58% identity with alpha9. Whereas alpha10 fails to produce functional receptors alone, it promoted robust acetylcholine-evoked currents when coinjected with alpha9. The presence of alpha10 modifies the physiological and pharmacological properties of the alpha9 receptor indicating that the two subunits coassemble in a single functional receptor. Fusing the N-terminal domain of alpha9 with the rest of the alpha10-cDNA yielded a functional alpha9:alpha10-chimera that displays the acetylcholine binding properties of alpha9 and ionic pore characteristics of alpha10-containing receptors. In addition, alpha9- and alpha10-subunit mRNAs show limited similar tissue distribution patterns and are expressed in cochlea, pituitary gland, and keratinocytes. These data suggest that, in vivo, alpha9-containing receptors coassemble with alpha10-subunit.