Retina from 1-day-old chicks is a valuable tissue model for studying neuronal nicotinic receptors because it expresses a large number of the developmentally regulated high affinity [(3)H]epibatidine labeled nicotinic receptors. Most of these receptors contain the beta4 subunit associated with different alpha subunits. Using a sequential immunodepletion procedure with anti-alpha6, anti-beta3, anti-beta2, and anti-beta4 antibodies, we purified an alpha4beta4 nicotinic receptor subtype that accounts for approximately 20 to 25% of the high affinity [(3)H]epibatidine labeled receptors present in retina at that developmental time. Immunoprecipitation and Western blotting experiments confirmed that the purified subtype contains only the alpha4 and beta4 subunits. This receptor binds a number of agonists and the antagonist dihydro-beta-erythroidine with nanomolar affinity, whereas it has micromolar affinity for the alpha-conotoxin MII and methyllycaconitine toxins and other nicotinic antagonists. Comparison of the pharmacological profile of this purified native subtype with that of the same subtype transiently expressed in human BOSC23 cells showed that they have very similar rank orders and absolute Ki values for several nicotinic drugs. Finally, because chick retina expresses an alpha6beta4-containing subtype with a high affinity for the alpha-conotoxin MII, we used native and transfected alpha4beta4 and alpha6beta4 subtypes to investigate the relative contributions of the alpha and beta subunits to this binding, and found that the alpha6 subunit determines the high affinity for this toxin.