The serotonin receptor type 3 is a pentameric ligand-gated ion channel regulating intestinal motility, nausea, and vomiting in humans. The HTR3B gene codes for the subunit B of this receptor. The HTR3B transcription start site is not unequivocally identified. In the present study we used transcription start site analyses, transcript-specific RT-PCR, and functional promoter analyses to identify the 5' structure of the HTR3B gene. According to these experiments, two alternative promoters control the expression of different HTR3B transcripts in the peripheral and central nervous system. The transcription start sites observed in the intestine corresponded to the current human genome annotation (NCBI Build 36.1, March 2006). The transcription start sites in the brain, however, were localized in a region about 4000 bp downstream. The brain transcripts lacked the coding first exon of the HTR3B structure published earlier but had an upstream-extended exon 2 containing a new potential translational start site. Reporter gene analyses showed significant promoter activity of the genomic region located 1560 bp upstream to 93 bp downstream of the brain-specific transcription start sites. This data suggests a different transcriptional regulation of the HTR3B gene in the peripheral and the central nervous system that leads to the expression of transcripts with variations in the 5' coding sequence. Further studies on the expression, structure and function of therefore expected tissue-specific 5-HT(3B) isoforms are required.