Recently, a murine paired-like homeobox gene, Phox2a, has been identified whose product is critical for the development of several major noradrenergic neuron populations, including the locus coeruleus. In noradrenergic neurons, dopamine beta-hydroxylase (DBH) is a hallmark protein and catalyzes the conversion of dopamine to noradrenaline. Our previous studies have shown that a composite promoter (domain IV), residing at -185 to -150 bp upstream of the transcription start site, is critical for DBH transcription and is comprised of multiple cis-acting elements, including a cyclic AMP response element, a YY1 binding site, and two core motifs of the homeodomain (HD)-binding site. Here, we show that the HD-binding site residing within domain IV is a noradrenergic-specific cis-acting element. In contrast, the cyclic AMP response element is active in all cell lines tested. We provide evidence that Phox2a is expressed only in DBH-positive cell lines and interacts with the HD-binding site. Forced expression of Phox2a robustly activates DBH promoter activity in DBH-negative cell lines (>10-fold), but increased it only marginally (<50%) in DBH-positive cell lines. Furthermore, another protein factor with an identical HD, Phox2b, also activates DBH transcription with an efficiency comparable to that of Phox2a. In contrast, neither Phox2a nor Phox2b was able to transactivate tyrosine hydroxylase transcription, indicating that these transcription factors differentially activate catecholamine-synthesizing gene transcription. Together with the Phox2a knockout experiment, the studies described here make Phox2a and Phox2b the first strong candidate transcription factors for determining a neurotransmitter phenotype in vertebrates.