Regulation of serotonin (5-HT)1A receptor expression in brain is implicated in mood disorders such as depression and anxiety. Transcriptional activity of the human 5-HT1A receptor gene was strongly repressed by a negative regulatory region containing a consensus repressor element-1 (RE-1) and two copies of the dual repressor element (DRE) identified in the rat 5-HT1A receptor gene. REST/NRSF, a silencer of neuronal genes, bound the 5-HT1A RE-1 and repressed the 5-HT1A promoter. Inactivation of RE-1 completely abolished REST-mediated repression, but resulted in only partial (15-50%) de-repression of basal 5-HT1A promoter activity. The human 5-HT1A DRE sequences bound specifically to the novel repressor Freud-1 (5'repressor element under dual repression binding protein-1) and conferred repressor activity at 5-HT1A or SV40 promoters. In 5-HT1A-negative cells [L6, human embryonic kidney (HEK) 293], the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) abolished repression mediated by both RE-1/REST and DRE/Freud-1, and induced almost complete de-repression of the 5-HT1A gene. By contrast, in 5-HT1A-expressing neuronal cells (RN46A, SN-48) TSA blocked RE-1/REST repression, but did not affect DRE/Freud-1-mediated repression. Thus in contrast to REST, Freud-1 mediates HDAC-independent repression of the 5-HT1A receptor promoter in neuronal 5-HT1A-positive cells, suggesting that HDAC recruitment might influence neuron-specific gene expression by further silencing expression in non-neuronal tissue.