Background: Conflicting associations with heroin dependence have been found involving the A1 allele of dopamine D2 receptor gene ( DRD2) TaqI A polymorphism. Methods: We compared two samples of unrelated Spanish individuals, all of European origin: 281 methadone-maintained heroin-dependent patients ( 207 males and 74 females) who frequently used non-opioid substances, and 145 control subjects ( 98 males and 47 females). Results: The A1-A1 genotype was detected in 7.1% of patients and 1.4% of controls ( P = 0.011, odds ratio = 5.48, 95% CI 1.26-23.78). Although the A1 allele was not associated with heroin dependence in the entire sample, the frequency of A1 allele was higher in male patients than in male controls ( 24.4% vs. 16.3%, P = 0.024, odds ratio = 1.65, 95% CI 1.07-2.57). A logistic regression analysis showed an interaction between DRD2 alleles and gender ( odds ratio = 1.77, 95% CI 1.15-2.70). Conclusion: Our results indicate that, in Spanish individuals, genotypes of the DRD2 TaqI A polymorphism contribute to variations in the risk of heroin dependence, while single alleles contribute only in males.