Department of Chemical Engineering, Biotechnology Division, University of Calcutta, 92, Acharya Prafulla Chandra Road, Kolkata-700 009, India Manuscript received 22 July 2009, revised 15 December 2009, accepted 18 December 2009 The objective or this investigation was to develop a new auxotrophic mutant from a regulatory mutant and to examine its potency for L-glutamic acid production. A high L-glutamic add yielding strain Micrococcus glutamicus AB100 was developed from a regulatory mutant Micrococcus glutamicus AB1 by subsequent mutagenic treatments with ethyleneimine solution and UV rays respectively. Micrococcus glutamicus AB1 was treated with ethylenelmlne solution, the auxotrophic mutants isolated show varied pattern of extracellular amino acids. Seventy auxotrophic strains were obtained, out of which only twenty seven excreted 0.5-3.0 mg, L-glutamic acid per ml and all auxotrophs require biotin for growth and production of amino acid. Sixteen auxotrophs produced 1.0-2.5 mg, L-glutarnic acid per ml and these auxotrophs required amino acids for their growth. Other auxotrophs lost their excretion capacity in subsequent fermentation trials. Further mutation of the biotin requiring auxotroph Micrococcus glutamricus AB10 with UV rays resulted In the isolation of seventy seven auxotrophic strains out of which only thirty excreted L-glutamic acid (up to 6.25 mg/ ml) higher than the parent auxotroph.