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Different Rhizobium species may be identified by using polymorphisms in their glutamine synthetases (GSII) but not by their GSI profiles. We analyzed the GSs of various Rhizobium tropici and Rhizobium etli strains (which are capable of nodulating and fixing nitrogen in Phaseolus vulgaris beans), as well as strains of other species included for comparison. The GS polymorphisms were determined by identifying variations in native enzyme mobility (revealed by GS activity staining) and in the isoelectric points of the monomers (revealed by immunodetection with antibodies against the GS proteins) by using gel electrophoresis. Restriction fragment length polymorphism patterns obtained by hybridizing an internal fragment of the GSII gene obtained from R. etli with total fragmented DNAs from different strains clearly distinguished the different groups. GSII is a novel and useful marker for Rhizobium groups and species, and GSII data support R. tropici and R. etli as bona fide species.