The regulatory properties of the enzymes involved in the aromatic amino acid biosynthesis of Pichia guilliermondii were investigated and compared with the regulatory pattern found in other yeast species. 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, anthranilate synthase, chorismate mutase and prephenate dehydrogenase are key regulatory enzymes in P. guilliermondii. Two distinctly regulated isozymes of DAHP synthase, the initial pathway enzyme, which is inhibited by tyrosine or phenylalanine were separated by DEAE-cellulose chromatography and were characterized. Tryptophan is an excellent feedback inhibitor of anthranilate synthase, the first definite step in tryptophan biosynthesis. There are two controlled enzymes within the specific synthesis of phenylalanine and tyrosine, chorismate mutase and prephenate dehydrogenase. Chorismate mutase exhibits a balanced allosteric responsivity to phenylalanine and tyrosine, when these are used as inhibitor; tryptophan acts as an allosteric activator. Tyrosine is an effective inhibitor of prephenate dehydrogenase, whereas the activity of prephenate dehydratase is not affected by any of the aromatic amino acids. The synthesis of the enzymes in the yeast was not repressed by any single exogenous aromatic amino acids, nor by combinations of the same.