Denitrification and nitrate ammonification are considered the highest-energy-yielding respiration systems in anoxic environments after oxygen has been consumed. The corresponding free energy changes are 7 and 35% lower than that of aerobic respiration, respectively. Growth yield determinations with pure cultures of Paracoccus denitrificans and Pseudomonas stutzeri revealed that far less energy is converted via ATP into cell mass than expected from the above calculations. Denitrification with formate or hydrogen as electron donor yielded about 2.4 to 3.0 g dry matter per mol formate or hydrogen and 15 to 18 g dry matter per mol acetate. Similar yields with acetate were obtained with Pseudomonas stutzeri. Wolinella succinogenes and Sulfurospirillum deleyianum, which reduce nitrate to ammonia, both exhibited similar yield values with formate or H 2 plus nitrate. The results indicate that ATP synthesis in denitrification is far lower than expected from the free energy changes and even lower than in nitrate ammonification. The results are discussed against the background of our present understanding of electron flow in denitrification and with respect to the importance of denitrification and nitrate ammonification in the environment.Oxidation of biomass proceeds preferentially with oxygen as electron acceptor, and only after its consumption in deeper sediment layers are alternative electron acceptors such as nitrate, manganese(IV), ferric iron, sulfate, or CO 2 reduced (15, 30). After oxygen reduction, reduction of nitrate to N 2 (denitrification) or to ammonia (nitrate ammonification) is the highest-energy-yielding process as shown by the following equations calculated with glucose as a representative of biomass (calculated based on the tables in reference 25):C 6 H 12 O 6 ϩ 6 O 2 3 6 CO 2 ϩ 6 H 2 O (⌬G°ЈϭϪ2,870 kJ per mol glucose) Even if we assume substrate and product concentrations more appropriate to natural conditions, i.e., 1 M glucose and 10 M nitrate and ammonium, the free energies of reactions 1 and 3 change by ϩ34 kJ and that of equation 2 changes by ϩ171 kJ, which still leaves a substantially higher amount of free energy with the denitrification process. From this comparison, one would expect that ATP synthesis as measured via growth yields in denitrification should come close to that of aerobic respiration, and yields in nitrate ammonification should be substantially lower. Thus, denitrification is expected to be the preferred process over nitrate ammonification, especially if the overall energy yield of a metabolic process is low. Contrary to this assumption, nitrate ammonification was reported to be the preferred process in anoxic environments in which the nitrate supply is limiting (26,27). Moreover, nearly all recently described lithotrophic bacteria oxidizing sulfide with nitrate as electron acceptor at neutral pH, e.g., Thioploca sp. or Thiomargarita sp., convert nitrate to ammonia (12,16,17), although denitrification should provide them with more energy. The energetics of nitrate reduction and th...