The metabolism of glutamine by a suspension of rat kidney tubules was studied in vitro. The influence of duration of incubation, glutamine concentration, and metabolic state of the donor animals was investigated. The relative importance of glucose synthesis, amino acid production, and oxidation to CO2 was estimated by drawing a complete balance of the nitrogens and the carbon chains of the extracted glutamine. It was found that the initial (first 15 min) rate of glutamine utilization was significantly greater than the subsequent rate due to an initial, but transient, extracellular accumulation of glutamate. This phenomenon was suppressed when a small amount of glutamate was added to the incubation medium. Glucose production constitutes the major fate for glutamine metabolism. No net oxidation of glutamine could be detected with 1 mM glutamine during the first 30 min. However, glutamine oxidation becomes significant after prolonged incubation (16% at 120 min). The metabolic fate of glutamine differs when 5 or 10 mM are presented to the tubules, glutamate production and oxidation to CO2 becoming more important. Metabolic acidosis or a 48-h fast increases glutamine extraction and enhances its utilization glucose synthesis while they depress glutamate accumulation and oxidation to CO2. Metabolic alkalosis has the opposite effect. It is concluded that the metabolism of glutamine in vitro is dependent on the conditions of the study. Furthermore, total oxidation to CO2 is not a major fate for glutamine metabolism at physiological concentration and is not enhanced by acidosis in the rat kidney in vitro.