Alternative oxidase (AOX) represents a non-energy conserving pathway within the mitochondrial electron transport chain. One potential physiological role of AOX could be to manage leaf carbohydrate amounts by supporting respiratory carbon oxidation reactions. In this study, several approaches tested the hypothesis that AOX1a gene expression in Nicotiana tabacum leaf is enhanced in conditions expected to promote an increased leaf carbohydrate status. These approaches included supplying leaves with exogenous carbohydrates, comparing plants grown at different atmospheric CO₂ concentrations, comparing sink leaves with source leaves, comparing plants with different ratios of source to sink activity, and examining gene expression over the diel cycle. In each case, the pattern of AOX1a gene expression was compared with that of other genes known to respond to carbohydrates and/or other factors related to source:sink activity. These included GPT1 and GPT3 (that encode chloroplast glucose 6-phosphate/phosphate translocators), SPS (that encodes sucrose phosphate synthase), SUT1 (that encodes a sucrose/H⁺ symporter involved in phloem loading) and UCP1 (that encodes a mitochondrial uncoupling protein). The AOX1a transcript amount was higher following the leaf sink-to-source transition, and in plants with higher source relative to sink activity due to increasing plant age. Further, these effects were amplified in plants grown at elevated CO₂ to stimulate source activity, particularly at end-of-day time periods. The AOX1a transcript amount was also higher following treatment of leaves with carbohydrate, in particular sucrose. Overall, the results provide evidence that, while source leaf sucrose accumulation may signal for a down-regulation of sucrose synthesis and transport, it also signals for means to manage the excess cytosolic carbohydrate pools. This includes increased AOX respiration to support carbon oxidation pathways even if energy charge is high, in combination perhaps with some return flux of carbohydrate from cytosol to stroma through the GPT3 translocator. As discussed, these activities could contribute to maintaining plant source:sink balance, as well as photosynthetic and phloem loading capacity.