Glutamine synthetase (GS), the enzyme responsible for the first step in the assimilation of ammonium in higher plants, generally exists in a number of isoforms associated with different tissues or cellular compartments within the plant. This study has investigated the GS isoenzyme composition of roots and N-fixing root nodules of the temperate legume, Lotus comiculatus, and has used a novel transgenic approach to manipulate the spatial distribution of the enzyme within these tissues. The isoforms of GS were studied using ion-exchange chromatography and western blotting, and a nodule-specific isoform was identified. Nitrate treatment of the N2-fixing plants had a marked effect on the nodule isoform, converting it to a form that was indistinguishable by ion-exchange chromatography from the root isoenzyme. To allow tissue-specific manipulation of GS activity, a chimaeric gene was constructed consisting of a translational fusion between the pat and uidA genes coding for phosphinothricin acetyl transferase (PAT) and β-glucuronidase (GUS), respectively. The PAT enzyme detoxifies the GS inhibitor, phosphinothricin, while GUS is a readily assayable marker enzyme that allowed the localisation of PAT activity within the plant tissues to be inferred. The pat::uidA gene was shown to encode a bifunctional enzyme when expressed in E. coli and in transgenic L. comiculatus plants. Transgenic lines carrying a nodule-specific promoter fused to pat::uidA were resistant to PPT only when nodulated. In some of these lines, nodule GS activity was completely resistant to soil applications of PPT under conditions where root GS was 100% inhibited. After long-term PPT treatment, one line (12E) showed a two-fold increase in nitrogenase activity, a four-fold increase in GS activity, and a 50% increase in dry matter production. Possible explanations for how specific inhibition of root GS led to these effects are discussed.