Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C4 dicot),Pisum sativum L. (C3 species) andParthenium hysterophorus L. (C3-C4. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C4 plant had a low specific activity, exhibited lower KM for glycolate, and required a lower pH for maximal activity, compared to the C3 enzyme. The enzyme from the C4 species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C3 plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C4 plant to α-hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C3 source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C4 plant,Amaranthus hypochondriacus are different from those of the C3 species or the C3-C4 intermediate.