PURPOSE Pegaspargase (PEG-ASP) has largely replaced native Escherichia coli asparaginase (L-ASP) in the treatment of acute lymphoblastic leukemia because of its longer half-life and lower immunogenicity. Risk factors for allergic reactions to PEG-ASP remain unclear. Here, we identify risk factors for reactions in a front-line acute lymphoblastic leukemia trial and assess the usefulness of serum antibodies for diagnosing allergy and predicting rechallenge outcome. PATIENTS AND METHODS PEG-ASP was administered to 598 patients in St Jude’s Total XVI study. Results were compared with Total XV study ( ClinicalTrials.gov identifiers: NCT00549848 and NCT00137111 ), which used native L-ASP. Serum samples (n = 5,369) were analyzed for anti–PEG-ASP immunoglobulin G by enzyme-linked immunosorbent assay. Positive samples were tested for anti–polyethylene glycol (PEG) and anti–L-ASP. We analyzed potential risk factors for reactions and associations between antibodies and reactions, rechallenge outcomes, and PEG-ASP pharmacokinetics. RESULTS Grade 2 to 4 reactions were less common in the Total XVI study with PEG-ASP (81 [13.5%] of 598) than in the Total XV study with L-ASP (169 [41.2%] of 410; P = 1.4 × 10⁻²³). For Total XVI, anti-PEG, not anti–L-ASP, was the predominant component of anti–PEG-ASP antibodies (96%). In a multivariable analysis, more intrathecal therapy (IT) predicted fewer reactions ( P = 2.4 × 10⁻⁵), which is consistent with an immunosuppressant contribution of IT. Anti–PEG-ASP was associated with accelerated drug clearance ( P = 5.0 × 10⁻⁶). Failure of rechallenge after initial reactions was associated with anti–PEG-ASP ( P = .0078) and was predicted by the occurrence of angioedema with first reaction ( P = .01). CONCLUSION Less IT therapy was the only independent clinical risk factor for reactions to PEG-ASP. PEG, and not L-ASP, is the major antigen that causes allergic reactions. Anti–PEG-ASP has utility in predicting and confirming clinical reactions to PEG-ASP as well as in identifying patients who are most likely to experience failure with rechallenge.