AbstractThe polymorphic 5′ upstream region of the dopamine D4 receptor (DRD4) gene containing several single nucleotide polymorphisms (SNPs) has recently become a focus of association studies in psychiatric genetics. Most SNP genotyping methods are based on the two‐step procedure of restriction fragment length polymorphism (RFLP). An alternative technique is a single‐step method of allele‐specific amplification (ASA), previously introduced for genotyping the −521 C/T SNP of the DRD4 promoter region and applied here for the −616 C/G SNP. Parallel genotyping of individuals with the novel ASA method and the conventionally used Ava II RFLP showed a potential underestimation of the −616 GG genotype frequency by the conventional method. Sequencing the dubious samples clearly demonstrated a novel A/G SNP at the −615th position influencing the Ava II digestion and thus resulting in misgenotyping. To avoid this problem, we introduced the Sau96 I RFLP for the −616 C/G genotyping as this restriction enzyme is not sensitive for the −615 A/G sequence variation. Allele (−616 G = 0.48; −616 C = 0.52) and genotype (−616 GG = 0.25; −616 GC = 0.46; −616 CC = 0.29) frequencies were determined by both the novel ASA and the Sau96 I methods. The obtained genotype frequencies corresponded to the Hardy–Weinberg equilibrium in our healthy Caucasian sample (N = 534, P = 0.168). Using these methods, no association was found between the −616 C/G SNP and personality factors of Cloninger's temperament and character inventory (N = 153) in our population. © 2003 Wiley‐Liss, Inc.