Anthranilate synthase from Euglena gracilis strain Z was purified to electrophoretic homogeneity. The enzyme migrated as a single symmetrical peak on sucrose density gradients with a sedimentation constant of about 5.0 S. The molecular weight determined by gel filtration on Sephadex G-200 was 80,000 +/- 2,500. Polyacrylamide gel electrophoresis of the pure enzyme after sodium dodecyl sulfate denaturation gave a single band at a position corresponding to a molecular weight of 79,000 +/- 2,000. These results suggest that Euglena antranilate synthase is composed of a single polypeptide chain of about 80,000 that possesses both ammonia- and glutamine-dependent activity. All other known glutamine-dependent anthranilate synthases are heterodimers or heterotetramers with the chorismic acid and glutamine binding sites on distinct polypeptide chains.