In order to reveal cold hardiness mechanisms and ascertain suitable cold hardiness biochemical indicators of walnut (Juglans regia), three walnut cultivars ‘Hartley’, ‘Jinlong 1’ and ‘Jinlong 2’ with strong to weak tolerance of chilling stress, were used to investigate variations of leaf antioxidant enzyme activity and superoxide anion (O₂^-_·) content in one yearold branches under chilling stress at 1 ℃ in leafexpansion period. The mesophyll cells ultrastructure of ‘Hartley’ and ‘Jinlong 2’ under chilling stress were also observed by transmission electron microscope. The results showed that the superoxide dismutase (SOD) and peroxidase (POD) enzyme activities were the strongest and O₂^-_· content was the lowest in chillingtolerant cultivar ‘Hartley’ under chilling stress among the three cultivars. The ultrastructure of the mesophyll cells was stable, and chilling injury symptoms of the leaves were not observed. In chillingsensitive cultivar ‘Jinlong 2’, the SOD, POD and catalase enzyme (CAT) activities decreased sharply, and the O₂^-_· content was kept at a high level under chilling stress. The ultrastructure of the mesophyll cells was injured obviously at 1 ℃ for 72 hours. Most of chloroplasts were swollen, and grana lamella became thinner and fewer. A number of chloroplasts envelope and plasma membrane were damaged and became indistinct. At the same time, the edges of some of ‘Jinlong 2’ young leaves became watersoaked. It was concluded that the ultrastructure stability of mesophyll cells under chilling stress was closely related to walnut cold hardiness. SOD, POD enzyme activities and O₂^-_· content in walnut leaves could be used as biochemical indicators of walnut cold hardiness in leafexpansion period. There might be a correlation between the damage of cell membrane system and reactive oxygen accumulation under chilling stress.