The Acyl CoA dependent oxidase 3 (Aox3p) from the yeast Yarrowia lipolytica, expressed in Escherichia coli, as an active protein with a 6 His tag at its N-terminal region has been purified to electrophoretic homogeneity. The purified enzyme exhibits a specific activity of 1.95 microM/min/mg using hexanoyl-CoA as substrate, and it remains active for at least 1 month upon storage at -30 degrees C in the presence of 35% (V/V) glycerol. The pH and temperature optima of the enzyme are 7.4 and 28-38 degrees C, respectively. Aox3p catalyzes the oxidation of both aliphatic acyl-CoA substrates of different chain lengths (e.g., hexanoyl-CoA, decanoyl-CoA, myristyl-CoA) as well as of the aromatic/heterocyclic ring-substituted chromogenic substrates, such as furylpropionyl-CoA. Of the above substrates, the efficiency of the enzyme, as judged by its kcat to Km ratio, exhibits the following order: decanoyl CoA > myristyl CoA > hexanoyl CoA > furyl-propionyl-CoA (FPCoA). Phenol, which is normally used in the coupled assay system for monitoring the H2O2 formation, functions as both an activator (at low concentrations) and a competitive inhibitor (at high concentrations) with respect to acyl-CoA substrates. The magnitude of activation and inhibition of the enzyme is dependent on the nature of the acyl-CoA substrates.