Previous studies, using ion-exchange chromatography, reported that one isoform of glutamine synthetase (GS) existed in the cytosol of rice (Oryza sativa L.) roots. We report that two isoforms of GS were observed in rice roots using native polyacrylamide gel electrophoresis (PAGE), GS activity staining and immunoblotting. One isoform (GSra) had a mobility similar to that of cytosolic GS in rice leaves (GSI). This isoform existed in seminal roots of germinating seeds and in the roots of seedlings grown in N-free and N-containing culture solutions. Another isoform (GSrb) moved more slowly than GSra and occurred only in the presence of external N. Both ammonium-N and nitrate-N induced GSrb, but the activity of GSrb was higher under ammonium-N than nitrate-N. After the removal of N source from the solution, GSrb activity and GSrb protein disappeared as judged by native-PAGE and immunoblotting but GSra remained relatively constant. It was estimated that GSrb contributed about 80% of total GS activity of the rice roots grown in the solution containing NH4+. SDS-PAGE and immunoblotting test showed that subunits of GSra, GSrb, and GS1 had the same molecular weight. Our results suggest that GSra was a constitutive enzyme and GSrb was an enzyme induced by external N.