l-asparaginase is a widely used cancer chemotherapy enzyme. The source for the enzyme with this property is mainly bacterial and its synthesis is strongly regulated by oxygen. In this study, we utilized two recombinant systems: one carried the gene (vgb) for the Vitreoscilla hemoglobin (VHb), a protein of prokaryotic origin which confers a highly efficient oxygen uptake to its host and the other carried the l-asparaginase gene (ansB). The host bacteria were Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Of these three bacteria, all gram-negative, E. coli and its recombinant strain showed up to sevenfold higher l-asparaginase activity in lactose than in other carbon sources. Although, in this bacterium glycerol was the poorest source for l-asparaginase synthesis, it supported the highest biomass production. In glucose medium, l-asparaginase activity of E. aerogenes was about threefold higher than its vgb and ansB recombinants. ansB recombinant showed significantly higher enzyme levels than both host and vgb recombinants in glycerol and lactose media. In this bacterium, VHb/vgb clearly caused a decrease in the enzyme synthesis under all conditions. As seen for E. aerogens, glycerol was the most favorable carbon source for P. aeruginosa and its vgb strain in terms of both l-asparaginase synthesis and biomass production. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol. Indeed, the highest biomass production for all bacteria and their recombinants was in glycerol. The VHb/vgb system is clearly advantageous for production of l-asparaginase in P. aeruginosa. The same, however, does not hold true for E. aerogenes.