Personality traits are among the most complex quantitative traits. Certain personality traits have been postulated to be part of the inherited component of substance dependence (SD) risk. Association between the M2 cholinergic receptor gene (CHRM2) and SD has recently been reported and replicated (Wang et al. Hum. Mol. Genet. (2004);13:1903-1911; Luo et al. Hum. Mol. Genet. 2005;14:2421-2434). In this study, we investigated the relationship between CHRM2 variation and personality traits in two American populations. We assessed dimensions of the five-factor model of personality, and genotyped six CHRM2 markers and 38 unlinked ancestry-informative markers in 239 subjects with SD [173 European-Americans (EAs) and 66 African-Americans (AAs)] and 275 healthy subjects (237 EAs and 38 AAs). The relationships between CHRM2 markers and personality traits were examined using multivariate analysis of covariance, controlling for marker-marker interaction effects and potential confounders. Associations were decomposed by Roy Bargmann stepdown analysis of covariance. Generally, substance-dependent patients, older individuals, males, and AAs scored higher on Neuroticism and lower on other personality factors. Diplotype CTCAAA/CTCGTT (P = 0.005) and the interaction between its two haplotypes (CTCAAA x CTCGTT) (P = 0.003) were associated with lower Conscientiousness scores. Haplotype CTCGAT (P = 0.006) and its interaction with haplotype TCAAAT (P = 0.002) were associated with higher Agreeableness scores. The trait-influencing variant site in CHRM2 for Agreeableness was close to marker rs1824024 (SNP3) (P = 0.002). CHRM2 variation may contribute to the genetic component of variation in personality traits. Personality traits might substantially underlie the heritable component of SD.