Homeodomain proteins play essential roles in basic processes during embryogenesis and development. NBPhox, a vertebrate paired-like homeodomain protein specific to catecholaminergic cells, may have a fundamental role in determining and maintaining the catecholaminergic phenotype. Here we describe the human and mouse genomic structures and the functional characterization of NBPhox. The genomic structure of NBPhox is highly conserved at the nucleotide level between human and mouse and is also quite similar to that of other paired-like homeodomain proteins, Arix/Phox2a, CRX, OTX1, and OTX2. The human NBPhox gene (HGMW-approved symbol PMX2B) maps to chromosomal region 5p12-p13. When the NBPhox expression plasmid was introduced into PC12h cells, the transcription from the promoter of the dopamine beta-hydroxylase (DBH) gene was slightly stimulated. However, NBPhox substantially enhances second messenger-mediated activation of the DBH promoter by forskolin and/or phorbol ester. Furthermore, we found that NBPhox can also enhance second messenger-mediated activation of the c-fos promoter and several enhancers, including cyclic AMP-response element, the binding site for activator protein 1, and serum-response element. Our findings provide strong evidence that a homeodomain protein is involved in the activation of several genetic regulatory elements responsive to second messenger-mediated signals. These data suggest that this family of proteins may be involved in determining and maintaining a cell-specific phenotype through regulation of certain genes responsive to second messenger-mediated signals.