The reasons why people smoke are varied, but research has shown that genetic influences on various aspects of nicotine addiction are a major factor. There also is a strong genetic influence on measures of nicotine sensitivity in mice. Despite the established contribution of genetics to nicotine sensitivity in mice and humans, no naturally occurring genetic variation has been identified that demonstrably alters sensitivity to nicotine in either species. However, one genetic variant has been implicated in altering nicotine sensitivity in mice is a T529A polymorphism in Chrna4, the gene that encodes the nicotinic receptor (nAChR) alpha4 subunit. The Chrna4 T529A polymorphism leads to a threonine to alanine substitution at position 529 of the alpha4 subunit. To more definitively address whether the Chrna4 T529A polymorphism does, in fact, influence sensitivity to nicotine, knock-in mice were generated in which the threonine codon at position 529 was mutated to an alanine codon. Compared with Chrna4 T529 littermate controls, the Chrna4 A529 knock-in mice exhibited greater sensitivity to the hypothermic effects of nicotine, reduced oral nicotine consumption and did not develop conditioned place preference to nicotine. The Chrna4 A529 knock-in mice also differed from T529 littermates for two parameters of acetylcholine-stimulated Rb+ efflux in midbrain: maximal efflux and the percentage of alpha4beta2* receptors with high sensitivity to activation by agonists. Results indicate that the polymorphism affects the function of midbrain alpha4beta2* nAChRs and contributes to individual differences in several behavioral and physiological responses to nicotine thought to be modulated by midbrain alpha4beta2* nAChRs.