Glutaminase-free l-asparaginase is known to be an excellent anticancer agent. In the present study, statistically based experimental designs were applied to maximize the production of glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428. Nine components of the medium were examined for their significance on the production of l-asparaginase using the Plackett-Burman experimental design. The medium components, viz., glucose, l-asparagine, KH2PO4, and MgSO4 center dot 7H(2)O, were screened based on their high confidence levels (P < 0.04). The optimum levels of glucose, l-asparagine, KH2PO4, and MgSO4 center dot 7H(2)O were found to be 2.076, 5.202, 1.773, and 0.373 g L-1, respectively, using the central composite experimental design. The maximum specific activity of l-asparaginase in the optimized medium was 27.88 U mg(-1) of protein, resulting in an overall 8.3-fold increase in the production compared to the unoptimized medium.