The conversion of -glutamate to -glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined. The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with -glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for -glutamate than -glutamate as a substrate. All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards -glutamate were much smaller than rates with the -isomer. These results are discussed in light of the observation that -glutamate is accumulated as an osmolyte in many archaea while -glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis.