Bacterial L-asparaginase has been widely used as therapeutic agent in the treatment of various lymphoblastic leukemia diseases. Studies on localization and production of novel glutaminase-free L-asparaginase were performed using Pectobacterium carotovorum MTCC 1428. The localization of L-asparaginase was carried out using cell fractionation techniques. The activity of L-asparaginase was found to be 85 and 77% in the cytoplasm of A carotovorum MTCC 1428 grown on medium containing L-asparagine and combination of L-asparagine and glucose respectively. Among the tested carbon sources, L-asparagine or the combination of L-asparagine and glucose was found to be the most suitable carbon sources to maximize the production of L-asparaginase. The maximum production of L-asparaginase was observed to be 14.56 U/ml (26.92 U/mg of protein) at 4 and 2 g/l of L-asparagine and glucose respectively. Yeast extract, L-asparagine and peptone have shown significant effect on the production of L-asparaginase. P. carotovorum MTCC 1428 has assimilated L-asparagine as an essential carbon source for maximizing the production of L-asparaginase. (C) 2009 Elsevier Ltd. All rights reserved.