Our objective was to investigate the relationship between the gene encoding the μ-opioid receptor (OPRM1) and susceptibility to substance dependence in European-American (EA) and African-American (AA) subjects. Eight single nucleotide polymorphisms (SNPs) at the OPRM1 locus, i.e., −2044C/A, −1793T/A, −1699insT, −1469T/C, −1320A/G, −111C/T, +17C/T (Ala6Val), and +118A/G (Asn40Asp) were genotyped in 676 subjects: 318 EA subjects and 124 AA subjects with substance dependence, and 179 EA normal controls, and 55 AA normal controls. Affection status was defined by each unique combination of alcohol, cocaine, and opioid dependence and analysis of association examined in relation to the possible combinations. We used a newly implemented permutation method to evaluate statistical significance. In EAs, a significant difference in haplotype frequency distributions was found between controls and “alcohol + opioid” dependent patients (P = 0.0036). This finding is also supported by logistic regression analysis and a simulation method. The frequencies of allele −2044A and haplotypes including −2044A are higher in these patients than in controls. In AAs, no allele, haplotype, or genotype frequencies were significantly different between cases and controls. There were highly significant differences in the allele, haplotype, and genotype frequencies between EA and AA controls. Four of the variants [−1793T/A, −1699insT, −1320A/G, and −111C/T] are in virtually complete linkage disequilibrium (LD) to compose a sequence pattern, which does not associate with any of the seven categories of substance dependence. In EAs, allele −2044A and haplotypes that include −2044A are the susceptibility allele and haplotypes for substance dependence. These findings suggest that OPRM1 may play a role in the pathophysiology of substance dependence and this role is population- and diagnosis-specific. © 2003 Wiley-Liss, Inc.