The capacity of rat neutrophils to utilize glutamine was investigated by 1) determination of oxygen consumption in the presence of glucose or glutamine, 2) measurement of maximal activity of phosphate-dependent glutaminase, 3) Northern blot, Western blot, and immunocytochemical detection of glutaminase, and 4) measurement of glutamine utilization and also production of ammonia, glutamate, aspartate, alanine, and lactate and decarboxylation of [U-14C]glutamine in cells incubated for 1 h. The rate of respiration by isolated neutrophils in the absence of added substrate was 5.0 nmol x min(-1) x 10(7) cells(-1). Maximal activity of phosphate-dependent glutaminase was 56 nmol x min(-1) x mg protein(-1) in freshly obtained neutrophils; the Michaelis-Menten constant was 3.5 mM for glutamine. This enzyme activity was inhibited by 2 mM glutamate, 2 mM oxoglutarate, and 2 mM NH4Cl. The presence of glutaminase protein (65 kDa) was confirmed by Western blot and immunocytochemical detection and the presence of the mRNA (6.0 kb) by Northern blot analysis. Glutamine was utilized by neutrophils incubated for 1 h at a rate of 12.8 nmol x min(-1) x mg protein(-1) when the amino acid was added to the medium at 2 mM, which is three to four times higher than the physiological concentration. In the presence of 0.5 mM glutamine, the amino acid was utilized at a rate of 2.9 nmol x min(-1) x mg protein(-1). The addition of 0.5 mM glutamate to the incubation medium caused a marked reduction (by 70%) in glutamine utilization by neutrophils. Glucose was utilized at 7.7 nmol x min(-1) x mg protein(-1) when cells were incubated in 5 mM glucose. The conversion of [U-14C]glutamine to 14CO2 was very low: <1% was totally oxidized. The formation of ammonia was approximately 27% of glutamine utilization, and the conversion of glutamine to glutamate, aspartate, alanine, and lactate accounted for approximately 84.6% of the total amino acid utilized by neutrophils. In this study, evidence is presented that, in addition to lymphocytes and macrophages, neutrophils also utilize glutamine.