Immunotoxins are fusion proteins of modified toxin conjugated to tumor cell selective ligand. Denileukin diftitox approved by FDA for treatment of CTCL is diphtheria toxin (DT)/IL2 fusion protein targeted to high affinity IL2R. Here, we have attempted to target the more uniquely expressed low affinity IL2R (IL2Rα). We designed four immunotoxins, SPRSV1 was designed to code for a single protein of DT (390) and IL2 (133) without any extra amino acids at the junction. SPRSV2 was designed to selectively target low affinity IL2R, it codes for DT (390) and IL2 (69). We also constructed SPRSV3 encoding for only DT (390) without any ligand, as negative control and SPRSV4 was designed similar to commercial equivalent denileukin diftitox, it codes for DT (387) and IL2 (133) with His at the junction. The cytotoxic activities of these immunotoxins were tested in various cell lines, cell lines lacking IL2R expression and healthy MNC were used as controls. The activities of SPRSV1 and SPRSV2 were comparable to that of SPRSV4. SPRSV2 exhibited potent cytotoxicity effectively targeted to α subunit of IL2R on various leukemia cell lines. Our studies also showed a negative correlation between CD25 expression and percentage cell viability after treatment with immunotoxins. © 2010 Elsevier B.V.