We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as α3, β2 and β4. In contrast, α6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the α6 subunit has long failed the critical test of functional reconstitution. Here we use patch-clamp techniques and confocal laser microscopy to measure ACh-activated currents and nicotine-elicited Ca2+ transients in human BOSC 23 cells transfected with chick α6 in combination with other chick nAChR neuronal subunits. Heterologously expressed α6 and β4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 μm). Likewise, ACh elicits measurable currents in cells transfected with α6 and β2. Hill analysis of the dose–response curves in cells transfected with α3, β4 and α6 cDNAs, suggests the assembly of functional α3β4α6 receptor, with an apparent affinity for ACh threefold lower than α3β4. Our results indicate that α6-containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.