The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), is an invasive and important pest in China. Understanding the physiological-biochemical characteristics of the overwintering pupae is essential for its cold tolerance and potential distribution in the northeast of China. Results indicated that the supercooling point (SCP) of H. cunea overwintering pupae varied significantly among Jilin city (43 degrees N, 126 degrees E), Jilin Province, Shenyang (41.8 degrees N, 123.4 degrees E), Liaoning Province and Yingkou (40.39N, 122.13E), Liaoning Province. The average SCP in Jilin was the lowest (-23.49 +/- 0.46 degrees C) in those of three areas. The water content in overwintering pupae had a significant positive correlation with the SCP (R = 0.848,P = 0.03). Contents of alanine (ALA) (26.00%), lysine (LYS) (30.51%) and arginine (ARG) (22.88%) in the overwintering pupae's hemolymph were higher than others. During the overwintering, the fat content in pupae's bodies decreased gradually. Analysis of variance (ANOVA) indicated that the content of trehalose in the body of H. cunea was remarkably different (P = 0.007) month by month, with the maximum in March (2.88 mg.mL(-1)) and the minimum (0.40 mg.mL(-1)) in November. Pearson's correlation test indicated that SCP had a significant negative correlation with the SOD enzyme activity (R = 0.850; P = 0.008). Thermal hysteresis activity (THA) of hemolymph was 0.68 degrees C and 0.7-0.8 degrees C for H. cunea pupae and larva, respectively, using differential scanning calorimetry, which suggests that the H. cunea can improve their cold tolerance through producing antifreeze proteins. These results could help us to predict the distribution of the H. cunea in the northeast of China. (C) 2014 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.