A callus line of manila grass [Zoysia matrella (L.) Merr.] has been maintained for 8 years in our laboratory. The present study investigated changes in ultrastructure and antioxidant enzyme activity during regeneration of the callus and examined the correlation between these changes and regeneration ability. The changes in fresh weight and diameter of the callus over time could be described by a sigmoidal growth curve with different stages. Electron microscopy revealed small embryonic callus cells, isodiametric in shape, with large, obvious nuclei, and dense cytoplasm. The cellular structures and morphology changed dramatically as regeneration proceeded. Of particular note was the formation of folded scutellum-like embryos at 14 d, which might be the turning point for morphological differentiation. Catalase (CAT) and peroxidase (POD) activities were the lowest at 14 d, the same time when superoxide dismutase (SOD) activity was highest. Thus, we speculate that the formation of the scutellum-like structures is associated with higher activity of SOD and lower activities of CAT and POD.