Interleukin 13 (IL13) binds a receptor that is highly overexpressed in malignant gliomas, IL13Ralpha2. IL13 protein is composed of four helices: alpha-helix A, B, C, and D, and we found a new "hot spot" in alpha-helix D that is crucial for the binding of IL13 to IL13Ralpha2. Lys-105 plus Lys-106 and Arg-109 represent this hot spot. In the current study, we have made substitutions at these three positions in IL13. We examined both neutralization of an IL13-based cytotoxin's glioma cell killing and direct receptor binding of the new IL13 mutants. We observed that Lys-105 and Arg-109 are critical for IL13 binding to IL13Ralpha2, indeed. However, new mutants of important properties were identified with regard to tumor targeting. IL13.K105R mutant, in which lysine was substituted by arginine, neutralized the killing of IL13Ralpha2-positive cells by IL13-based cytotoxin more efficiently than wild-type IL13. However, IL13.K105L or IL13.K105A was deprived of any such activity. Furthermore, IL13.K105R and IL13.R109K competed 77- and 27-fold better, respectively, with the binding of [(125)I]IL13 to the IL13Ralpha2 binding sites when compared with wild-type IL13. Thus, we have uncovered the first forms of IL13 of higher avidity toward IL13Ralpha2. These mutants should prove useful in the further design of anticancer diagnostics/therapeutics.