Eleven single-nucleotide polymorphisms (SNPs) spanning OPRD1 were examined in 1063 European Americans (EAs) (620 cases with substance dependence (SD), including 557 with alcohol dependence (AD), 225 with cocaine dependence (CD) and 111 with opioid dependence (OD), and 443 controls). Nominally significant associations (P<0.05) of five SNPs with SD were observed; only the association of the non-synonymous variant G80T with OD remained significant after correction for multiple testing using SNPSpD. Haplotype analyses with six tag SNPs indicated that a specific haplotype GCAACT, which harbors G80T G-allele and C921T C-allele, was significantly associated with AD (chi(2)=14.82, degrees of freedom (d.f.)=1, P<0.001), CD (chi(2)=9.19, d.f.=1, P=0.002) and OD (chi(2)=20.68, d.f.=1, P<0.001). Logistic regression analyses, with sex and age being considered, demonstrated that this haplotype had a risk effect on AD (P=0.03, beta=1.86, odds ratio (OR)=6.43) and especially on OD (P<0.001, beta=3.92, OR=50.57). Moreover, seven SNPs covering OPRK1 were examined in the majority of the above subjects (390 cases, including 327 AD, 177 CD and 97 OD subjects, and 358 controls). Although no significant differences in allele, genotype or haplotype frequency distributions were seen between cases and controls, a specific OPRK1 haplotype, GGCTTCT, was significantly associated with AD (chi(2)=8.12, d.f.=1, P=0.004). Logistic regression analyses also revealed its risk effect on AD (P=0.009, beta=1.06, OR=2.90). Population stratification artifact was not observed in the sample. Taken together, our findings supported a positive association between OPRD1 variants and SD, and a positive haplotypic association between OPRK1 and AD in EAs.