The closely related homeodomain transcription factors, Phox2a and Phox2b, are restrictively expressed in central and peripheral noradrenergic (NA) neurons in an overlapping but distinct manner, and critically regulate the differentiation and neurotransmitter identity of NA neurons. The structure and function of the human Phox2a (hPhox2a) promoter has recently been reported. Towards the long-term goal of delineating the regulatory cascade of NA neuron differentiation, we isolated a human Phox2b (hPhox2b) genomic clone encompassing approximately 7.8 kb of the 5' upstream promoter region, the entire exon-intron structure and 4.5 kb of the 3' flanking region. Two transcription start sites are identified to reside 115 and 110 nucleotides upstream of the start codon, based on both primer extension and 5'-rapid amplification of the cDNA ends analyses. In addition, transient transfection assays indicate that 1.1 kb or longer upstream sequences of the hPhox2b gene may confer cell type-specific gene expression in certain, but not all cell lines. The promoter activity of the hPhox2b gene is modestly transactivated by forced co-expression of Phox2b and the hPhox2b gene promoter contains a high-affinity binding site at -320 to -295 bp. This study provides a frame to further elucidate the molecular mechanisms underlying the regulation of Phox2a and Phox2b gene expression and its relation to NA differentiation.