Asparaginase (ASP) is a standard component of the antileukemia armamentarium. There are currently 3 preparations of asparaginase available: (1) E. coli (ASP, Elspar™); (2) the enzyme derived from Erwinia chrysanthemi (ERW, Erwinase™); (3) pegaspargase (PEG, Oncaspar™), the E. coli enzyme modified by covalent attachment of polyethylene glycol. This report describes the findings of 3 pharmacologic end points: ASP enzyme activity in patients’ sera, depletion of asparagine and the development of anti-ASP antibodies. Pharmacokinetics and pharmacodynamic studies in a group of naive children with newly diagnosed ALL demonstrate a significant difference in apparent half-life (1.24 days E. coli vs. 0.65 ERW vs. 5.73 PEG; p < 0.001) and days of asparagine depletion (14–23 E. coli vs. 7–15 ERW vs. 26–34 PEG; p < 0.01) for the 3 different preparations. Data from Pediatric Oncology Group (POG) Protocol #8866 show that high antibody levels correlated with rapid ASP clearance and a significantly lower response rate (NR = 26% vs. CR + PR+64%). The pharmacologie characteristics of ASP in terms of clearance of enzyme activity and ability to deplete serum asparagine was dependent upon the nature of the enzyme and are significantly altered in patients who develop anti-ASP antibodies regardless of their clinical status. In addition, these data demonstrate that ASP pharmacokinetics are directly related to its anti-leukemic effect. In order to maximize the therapeutic benefits of ASP, the optimal dose and schedule of treatment should be determined based on pharmacologic testing rather than by clinical criteria alone. Future studies will focus on the role of “silent hypersensitivity” as a mechanism of resistance to ASP and strategies to maximize the therapeutic efficacy of ASP as part of ALL therapy.