When hepatocytes suspensions obtained from whole livers of 48-h-fasted rats were incubated in Krebs-Henseleit buffer with a near-physiological concentration (1 mM) of L-[1-14C]glutamine as substrate, the apparent removal of glutamine was low, but the release of 14CO2 was much larger than the enzymatically measured removal of glutamine. This indicates that glutamine was metabolized at rates much higher than those accounted for by the apparent removal of glutamine. This also suggests that glutamine utilization was, at least in part, masked by concomitant synthesis of glutamine from endogenous substrates via glutamine synthetase. Evidence that such synthesis occurred was obtained by: (i) addition of methionine sulfoximine, an inhibitor of glutamine synthetase, which caused a large increase in the apparent removal of glutamine; and (ii) measurement of the specific radioactivity of L-[1-14C]glutamine which was shown to decrease during incubation. Addition of vasopressin (10(-7) M) led to a marked increase in glutamine removal by a dual mechanism: it accelerated flux through glutaminase, the enzyme which initiates the hepatic degradation of glutamine, and inhibited flux through glutamine synthetase.