L-Asparaginase is an important component in the treatment of acute lymphoblastic leukemia in children. Its antineoplastic activity toward malignant cells is due to their characteristic nature in slow synthesis of L-asparagine (Asn), which causes starvation for this amino acid, while normal cells are protected from Asn starvation due to their ability to produce this amino acid. The relative selectivity with regard to the metabolism of malignant cells forces to look for novel asparaginase with little glutaminase-producing systems compared to existing enzyme. In this investigation, the role of the extracellular asparaginase enzyme produced by an isolated bacterial strain was studied. Biochemical characterization denoted that this isolated bacterial strain belongs to the Bacillus circulans species. The strain was tested for L-asparaginase production, and it was observed that, under an optimized environment, this isolate produces a maximum of 85 IU ml(-1) within 24-h incubation. This enzyme showed less (60%) glutaminase activity compared to commercial Erwinia sp. L-asparaginase. The partially purified enzyme showed an approximate molecular weight of 140 kDa. This enzyme potency in terms of antineoplastic activity was analyzed against the cancer cells, CCRF-CEM. Flow cytometry experiments indicated an increase of sub-G1 cell population when the cells were treated with L-asparaginase.