AbstractThe 3′‐untranslated region (UTR) of the human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) domain, which is thought to be associated with dopamine‐related psychiatric disorders, personality, and behavior. However, the molecular and neuronal functions of polymorphisms within the VNTR domain are unknown. We previously identified the transcription factor HESR1 (HEY1) as a VNTR‐binding protein. Hesr1 knockout mice exhibit DAT up‐regulation in the brain and low levels of spontaneous activity. Other members of the HESR (HEY) family, including HESR2 (HEY2) and 3 (HEYL), have similar DNA‐binding domains. In this study, we analyzed the effects of HESR1, ‐2, and ‐3 on DAT1 expression in human neuroblastoma SH‐SY5Y cells using luciferase reporter assays. We found that the VNTR domain played an inhibitory role in DAT1 reporter gene expression and that HESR1 and ‐2 inhibited expression via both the core promoter and the VNTR. The inhibitory effects of HESR family members on DAT reporter gene expression differed depending on the number of repeats in the VNTR domain. We also found that each Hesr was expressed in the dopaminergic neurons in the mouse midbrain. These results suggest that the HESR family is involved in DAT expression via the VNTR domain. © 2011 Wiley‐Liss, Inc.