Previously, a missense polymorphism was identified in the mouse nicotinic receptor α4 subunit gene, Chrna4. This polymorphism leads to an Ala/Thr variation at amino acid position 529 of the α4 subunit. Chrna4 A529T is associated with several measures of acute sensitivity to nicotine as well as with mouse strain differences in nicotine-stimulated 8 6 Rb + efflux from synaptosomes. Here, we report that the variant forms of the mouse α4 subunit confer functional differences when expressed with the β2 subunit in a heterologous system. α4β2 receptors containing the T529 variant of the α4 subunit exhibited a higher EC 5 0 value for the high affinity receptor population and an apparent reduced sensitivity to blockade by DHβE relative to α4β2 receptors containing the A529 variant of the α4 subunit. Moreover, the proportion of the total agonist-elicited current contributed by the high affinity α4β2 receptor population was greater for α4β2 receptors containing the α4(T529) variant (64%) than the α4β2 receptors containing the α4(A529) variant (41%). These data suggest that the polymorphism in the mouse α4 subunit is located in a previously unidentified functional domain of the receptor subunit that influences receptor function, including regulation of the affinity population ratio of α4β2 receptors.