We tested the hypotheses that relative activity of the less efficient alternative oxidase (AOX) path changes with diurnal temperature changes, and thus changes carbon use efficiency with temperature. The activities of the alternative and cytochrome oxidase (COX) paths in plant tissues of three species were determined by measuring 18O/16O discrimination and total respiration from 17 to 36 degrees C. A new, more accurate method for calculating oxygen uptake rate from the mass spectrometry data was developed. Total carbon use efficiency was calculated from the ratio of respiratory heat and CO2 rates measured from 10 to 35 degrees C. Oxygen isotope discrimination (22.9 +/- 0.4 per thousand) and AOX participation were invariant with temperature in leaf tissue of Cucurbita pepo, Nicotiana sativa and Vicia faba, thus falsifying the first part of the hypothesis. Stress responses of respiration at the temperature extremes limited the range for which carbon use efficiency could be accurately measured to 15-30 degrees C in N. sativa, to 10-25 degrees C in C. pepo and to 20-30 degrees C in V. faba. Carbon-use efficiency was invariant at these temperatures in these species, demonstrating that changes in other pathways that would vary carbon-use efficiency were also invariant with temperature.