Experiments were undertaken to explain the occurrence of a high denitrification capacity in anaerobic, NO(3)-free habitats. Deep layers of freshwater sediments that were buried more than 40 years ago and digested sludge were the habitats studied. The denitrifier populations were 3.1 x 10 and 3.1 x 10 cells cm in deep sediments from a river and lake, respectively, and 5.3 x 10 cells cm in digested sludge. The denitrification capacities of the samples reflected the population densities. Strict anaerobic procedures were used to obtain the predominant isolates that would grow on anaerobic medium with NO(3). All strict anaerobes isolated failed to denitrify. All isolates that denitrified were aerobic, gram-negative bacteria, particularly species of Pseudomonas and Alcaligenes. No detectable growth was observed when these strains were incubated with electron acceptors other than NO(3) or O(2). When representative isolates were added to sterile, O(2)- and NO(3)-free porewater from their original locations at their natural densities (10 cells cm), no change in viable population was noted over 3 months of incubation. Metabolic activity was demonstrated in these cells by slow formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [C]glucose was added to cell suspensions of the pseudomonads that had been starved for 3 months without electron acceptors (O(2) or NO(3)), C-labeled products, including cell biomass, CO(2), and fermentation products, were produced. The high denitrification capacity of these anaerobic environments appears to be due to conventional respiratory denitrifiers. These organisms have the capacity for long-term survival without O(2) or NO(3) and appear to be capable of providing for their maintenance by carrying on a low level of fermentation.