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Knowledge of root respiration is a prerequisite for a better understanding of ecosystem carbon budget and carbon allocation. However, there are not many relevant data in the literature on direct measurements of in situ root respiration by root chamber method. Furthermore, few studies have been focused on the effects of root diameter (Dr) and root nitrogen concentration (Nr) on in situ root respiration among different seasons and tree species. To address these goals, we used a simplified root-chamber system to measure in situ root respiration rates of Acacia crassicarpa and Eucalyptus urophylla in subtropical plantations of south China. We found that the species and season variation in root respiration were affected by Dr and Nr. Also, the root respiration per unit dry mass (Rr, nmol CO2 g-1 s-1) and root respiration per unit N (Rn, nmol CO2 g N-1 s-1) were affected by Dr and Nr. The Rr, Rn, Nr and soil temperature sensitivity (Q10) of Rr for the two species significantly decreased with an increase of Dr. The Rr of the two species showed significant an inter-seasonal and diurnal pattern, and this trend decreased with increasing Dr. Both the Rr and Q10 of the two species increased with increasing Nr. The Dr and Nr explained 54 and 52% of the observed variation in Rr for A. crassicarpa, and 65 and 70% for E. urophylla. The Rr, Nr, and Q10 of A. crassicarpa were significantly higher than those of E. urophylla. Our results indicated that root respiration was dependent on Dr and Nr, and this dependence varied with season and plant species.