Indirect calorimetry is the method by which the type and rate of substrate utilization, and energy metabolism are estimated in vivo starting from gas exchange measurements. This technique provides unique information, is noninvasive, and can be advantageously combined with other experimental methods to investigate numerous aspects of nutrient assimilation, thermogenesis, the energetics of physical exercise, and the pathogenesis of metabolic diseases. Since its use as a research tool in metabolism is growing, the theoretical bases of indirect calorimetry are here reviewed in a detailed and orderly fashion. Special cases, such as the occurrence of net lipid synthesis or gluconeogenesis, are formally considered with derivation of explicit stoichiometric equations. The limitations of indirect calorimetry, both theoretical and technical, are discussed in the context of circumstances of clinical interest in metabolism.