Dopamine D4 receptor (DRD4) and serotonin transporter (SERT) gene polymorphisms were studied, as possible genetic risk factors for substance dependence. The case-control study involved a large cohort (n = 362) of healthy Caucasian population, and an initial sample of 73 substance dependent patients (including a subgroup of 53 heroin dependents). Improved methods were applied for genotype detection of the DRD4 polymorphisms (exon 3 48 bp VNTR; -521 C/T SNP and 120 bp duplication in the 5' flanking region) and the SERT gene polymorphisms (5-hydroxytriptamin transporter linked polymorphic region [5-HTTLPR] in the 5' flanking region and the intron 2 VNTR [STin2]). Association between the -521 C/T SNP of the DRD4 promoter region and substance dependence was significant in the subgroup of heroin dependents (p = 0.044). The other analyzed polymorphisms did not show any significant association, but an interaction between -521 C/T SNP of DRD4 and the 5-HTTLPR polymorphisms was observed. Association between the -521 CC vs. CT or TT genotypes and heroin dependence was enhanced in the presence of short (s or 14-repeat) 5-HTTLPR allele (p 0.01). The odds ratio of 2.14 observed for the -521 CC genotype increased to 4.82 in double homozygotes of -521 CC and 5-HTTLPR ss, emphasizing the importance of combined analysis of polymorphisms in the dopaminergic and serotonergic systems in heroin dependence. However, due to the limited size of our sample these results should be interpreted with caution.