Philodendron bipinnatifidum inflorescences heat up to 42 degrees C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX). Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses. Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90 of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O(2) isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O(2). The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O(2) can aid measurement of respiratory pathway fluxes in dense tissues.