Asparaginase (ASP) is used routinely in frontline clinical trials for the treatment of childhood acute lymphoblastic leukemia (ALL). The goals of this study were to assess the pharmacokinetics and pharmacodynamics of ASP and to mathematically model the dynamics between ASP and asparagine (ASN) in relapsed ALL. Forty children were randomized to receive either native or polyethylene glycolated (PEG) Escherichia coli ASP during reinduction therapy. Serial plasma ASP and ASN, cerebrospinal fluid (CSF) ASN, and serum anti-ASP antibody samples were collected. The ASP clearance was higher (P = 0.001) for native vs. PEG ASP. Patients with antibodies to PEG ASP had faster PEG ASP clearance (P = 0.004) than did antibody-negative patients. Patients who were positive for antibodies had higher CSF ASN concentrations than did those who were negative (P = 0.04). The modeling suggests that by modifying dosages, comparable ASN depletion is achievable with both preparations. At relapse, there were significant pharmacokinetic and pharmacodynamic differences attributable to ASP preparation and antibody status.