Carbon dioxide production in free living animals and humans can be measured using tracer techniques, but the prediction of energy expenditure also requires an estimate of the energy equivalents of CO2 (energy expended/CO2 produced; Eeq(CO2)). This work is concerned with assessing the variation in Eeq(CO2) with the use of dietary information, indirect calorimetry, and theoretical concepts. The Eeq(CO2) for diets (Eeq(CO2diet)) ingested by 63 individuals living in a Cambridgeshire village, UK, was found to vary by < 10%. The Eeq(CO2diet) for different populations varied by > 10% and for artificial enteral feeds by approximately 20%. Alcohol increases this variability because it has a particularly high Eeq(CO2). Variation in the nitrogenous end products of metabolism may also have a substantial effect on the Eeq(CO2) for a subject (Eeq(CO2body)), especially when a large proportion of energy expenditure is derived from protein oxidation, as in strict carnivores. Nutrient/energy imbalances such as those associated with growth, hypercaloric feeding, or starvation may also have major effects on Eeq(CO2body). It is concluded that the calculation of energy expenditure from CO2 production should not employ a universal value for Eeq(CO2body). The value should take into account the physiological and clinical state under investigation. Practical recommendations are suggested.