Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [13N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [13N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [13N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [13N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [15O]H2O. The reduction of [13N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [13N]ammonia may enable measurement of GS activity in the living brain. Synapse 69:26–32, 2015. © 2014 Wiley Periodicals, Inc.