The relationships between heat production, alternative oxidase (AOX) pathway flux, AOX protein, and carbohydrates during floral development in Nelumbo nucifera (Gaertn.) were investigated. Three distinct physiological phases were identified: pre-thermogenic, thermogenic, and post-thermogenic. The shift to thermogenic activity was associated with a rapid, 10-fold increase in AOX protein. Similarly, a rapid decrease in AOX protein occurred post-thermogenesis. This synchronicity between AOX protein and thermogenic activity contrasts with other thermogenic plants where AOX protein increases some days prior to heating. AOX protein in thermogenic receptacles was significantly higher than in post-thermogenic and leaf tissues. Stable oxygen isotope measurements confirmed that the increased respiratory flux supporting thermogenesis was largely via the AOX, with little or no contribution from the cytochrome oxidase pathway. During the thermogenic phase, no significant relationship was found between AOX protein content and either heating or AOX flux, suggesting that regulation is likely to be post-translational. Further, no evidence of substrate limitation was found; starch accumulated during the early stages of floral development, peaking in thermogenic receptacles, before declining by 89% in post-thermogenic receptacles. Whilst coarse regulation of AOX flux occurs via protein synthesis, the ability to thermoregulate probably involves precise regulation of AOX protein, most probably by effectors such as a-keto acids.