Background The human dopamine D4 receptor (DRD4) gene has been studied extensively as a candidate gene for certain psychological traits and several behavioural and psychiatric disorders. Both the 5' regulatory region and the coding sequence contain a number of polymorphisms. The promoter variants have received particular attention in the past few years due to their possible role in the regulation of gene transcription. Previously, the -521C/T SNP was shown to influence promoter activity. The aim of this study is to perform an in-depth analysis of this effect in the context of various neural cell lines. Results Endogenous mRNA expression of the DRD4 gene was demonstrated in two neuroblastoma (SK-N-F1, IMR32) and one retinoblastoma cell line (Y79) by RT-PCR. In addition, very low DRD4 mRNA levels were also detected in HeLa cells. The transcriptional activity of a series of 5' promoter deletion mutants was determined by transient transfection of luciferase reporter constructs. The activity profile of these promoter fragments was similar in each of the cell lines tested. The highest luciferase reporter activity was obtained with a construct containing promoter sequences between nucleotides -668 to -389, while a putative silencer region was localised spanning from nucleotide -1571 to -800. Surprisingly, the -521 C/T polymorphism had no significant effect on transcriptional activity of the reporter construct with the highest activity (-668 to -389) in any of the three cell lines tested. Conclusion Our results do not confirm previous data assigning different transcriptional activities to the -521 C/T alleles of the human DRD4 promoter. Furthermore, these findings highlight the need for further characterization of the 5' regulatory region of the DRD4 gene and identification of additional functional promoter polymorphic sites, especially in the context of haplotype.