By using a modification of the microtiter solid-phase radioimmunoassay, we have measuredEscherichia coliL-Asparaginase (L-ASP) specific IgG, IgG4, and IgE antibodies in children who received L-ASP as part of their chemotherapy for leukemia and lymphoma. In 13 children with acute lymphoblastic leukemia induced with vincristine, prednisone, and L-ASP (10,000 IU/M2i.v. each week for 3 weeks), seven developed high titer specific IgG antibodies. Four of the seven relapsed at the time of their peaking IgG response (6–10 months). None of the six with low or absent L-ASP antibody response have relapsed (followed for 20–35 months). In six children with allergic reactions to L-ASP reinduction, all had high titers of L-ASP specific IgG4(≥20 U/ml) at the time of their reaction. In 16 other children with low L-ASP IgG4(<13 U/ml), none demonstrated allergic reactions to rechallenge. Specific IgE was not consistently detectable in either group. In 21 patients with leukemia or lymphoma on L-ASP with cyclophosphamide-containing regimens, none developed significant IgG antibody response, compared with seven of 13 not receiving cyclophosphamide (p < 0.001). We conclude: (a) development of L-ASP antibodies may have prognostic significance; (b) the detection of specific IgG4can predict L-ASP allergy; and (c) cyclophosphamide-containing regimens reduce antibody formation to L-ASP and may allow repetitive (without anaphylaxis) and more effective (avoiding neutralizing antibodies) use of L-ASP.