Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates. We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J. (1994) J. Biol. Chem. 269, 26559 -26567). Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis. The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain. We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics. The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography. The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate. However, the two enzymes differ in many of their properties. DTH II has a lower K m value for the cosubstrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated. The two isoforms also have a different pH profile. As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels. However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition. The central nervous system isoform DTH I is activated through phosphorylation by cAMPdependent protein kinase (PKA) in the absence of dopamine. In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine. Site-directed mutagenesis of Ser 32 , a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA. We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects.Tyrosine hydroxylase (TH 1 ) (tyrosine 3-monooxygenase, EC 1.14.16.2) is an eukaryotic enzyme catalyzing the first and rate-limiting step in dopamine and other catecholamine biosynthesis, i.e. the hydroxylation of the monophenol amino acid L-tyrosine to produce the ortho-diphenol L-dihydroxyphenylalanine (2, 3). The enzyme is active in the presence of ferrous iron, O 2 , and a tetrahydrobiopterin co-substrate. A single gene encodes TH, which is required for embryonic development and survival in mammals (4, 5). In vertebrates, TH activity is exquisitely regulated at each step of its expression: control of gene transcription, RNA alternative processing, mRNA stability, and direct modulation of the enzyme by catecholamine feedback inhibition and protein kinase activation (6 -8).In contrast, much less is known on the regulatory properties of tyrosine hydroxylase in insects. Muta...