The gene encoding the salt-tolerant glutaminase I from marine Micrococcus luteus K-3 (M. luteus K-3) was cloned in Escherichia coli (E. coli) JM109. Clones were screened by hybridization with degenerate oligonucleotide probes designed using the known N-terminal amino acid sequence of glutaminase I from M. luteus K-3. A 2.4-kb HincII fragment from a 8-kb primary cloned DNA fragment was subcloned and sequenced. This fragment had an open reading frame of 1,368 nucleotides encoding 456 amino acids. The molecular weight of the deduced amino acid sequence of glutaminase I was determined to be 48,247. Comparison of the amino acid sequence of glutaminase I with those of kidney, brain, liver, and Caenorhabditis elegans glutaminases revealed high degrees of homology in several local regions.