Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 is expressed predominantly in the central nervous system whereas tryptophan hydroxylase 1 is mostly expressed in peripheral tissues. While the two enzymes share considerable sequence homology, the regulatory domain of tryptophan hydroxylase 2 contains an additional 41 amino acids at the N-terminus that tryptophan hydroxylase 1 lacks. Here we show that the extended tryptophan hydroxylase 2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, tryptophan hydroxylase 2 is synthesized less efficiently and is also less stable than tryptophan hydroxylase 1. Removal of the unique portion of the N-terminus of tryptophan hydroxylase 2 results in expression of the enzyme at a level similar to that of tryptophan hydroxylase 1 while protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10-20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased tryptophan hydroxylase 2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to tryptophan hydroxylase 2, these data provide evidence for selective regulation of brain serotonin synthesis.