Mild trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34 kDa fragment which is catalytically active. To determine the structure of the trypsin-digested tyrosine hydroxylase (tTH) relative to the native enzyme and to regulatory phosphorylation sites, bovine adrenal tTH was purified to homogeneity and the sequence of 17 amino acids from the N-terminus was determined. These data indicate that the N-terminus of tTH corresponds to amino acid 158. Thus the catalytic region is contained within the central region of enzyme approximately 17 kDa from the N-terminal and 5 kDa from the C-terminal and does not include phosphorylation sites located in the N-terminus. This region of TH shares a high degree of homology with phenylalanine hydroxylase and tryptophan hydroxylase and thus reflects a selective conservation of regions required for catalysis in contrast to the non-homologous regulatory sites. Activation by proteolysis corresponds to an increase in affinity for both substrate and cofactor indicating that the region removed by proteolysis imposes additional constraints on substrate and cofactor binding. These data are consistent with the model that the catalytic core of TH is contained within a 34 kDa region in the highly conserved central portion of the molecule whereas the non-homologous N-terminus regulates cofactor binding and directs substrate specificity. © 1988 Academic Press, Inc.