In Drosophila melanogaster, serotonin (5-hydroxytryptamine, 5-HT) is required for both very early non-neuronal developmental events, and in the CNS as a neurotransmitter to modulate behavior. 5-HT is synthesized, at least in part, by the actions of Drosophila tryptophan-phenylalanine hydroxylase (DTPH), a dual function enzyme that hydroxylates both phenylalanine and tryptophan. DTPH is expressed in numerous tissues as well as dopaminergic and serotonergic neurons, but it does not necessarily function as both enzymes in these tissues. Deficiencies in DTPH could affect the production of dopamine and serotonin, and thus dopaminergic and serotonergic signaling pathways. In this paper, we show that DTPH exhibits differential hydroxylase activity based solely on substrate. When DTPH uses phenylalanine as a substrate, regulatory control (end product inhibition, decreased PAH activity following phosphorylation, catecholamine inhibition) is observed that is not seen when the enzyme uses tryptophan as a substrate. These studies suggest that regulation of DTPH enzymatic activity occurs, at least in part, through the actions of its substrate.