Abstract: Effects of different low temperature stresses (14℃ and 4℃) on cyanide-resistant alternative pathway development and operation, as well as on alternative oxidase expression in plant calli of tobacco (Nicotiana rustica L.) were compared. The results showed that both alternative pathway capacity (Valt) and its actual operation activity (rValt) were dramatically induced under the two different low temperature stresses, and the change patterns of Valt and rValt were similar: both of them were induced at the early stage of stress treatment, the highest was found on 3 d and then declined to a relatively constant level. The increase of Valt was in relation to the temperature decreased, while no significant difference of rValt induced between 14℃ and 4℃. This suggests that responses of Valt and rValt to different temperature stress are different. The immunoblot results indicated that low temperature stress remarkably induced the content of total alternative oxidase (AOX) protein, and the changing mode throughout the stress was similar to Valt. The results of variation in AOX monomer and homodimer contents under stress treatment showed that AOX is largely located in mitochondria membrane of tobacco callus as the oxidized form (homodimer), and the existing state could not be changed by low temperature stress. The amount of monomer and homodimer both induced by low temperature stress, but no appreciable differences in the content of monomer was observed between two different low temperatures, and the level of homodimer induced under 4℃ was higher than that under 14℃. Our results suggest that, effects of low temperature on the development of cyanide-resistant alternative pathway are mainly due to the differently induced content of AOX homodimer under different low temperatures. The content of highly active AOX monomer form is not obviously induced by aggravating low temperature stress, thus the operation degree of cyanideresistant alternative pathway shows no notable difference between two kinds of low temperatures tested.