A fusion protein linking a truncated form of diphtheria toxin (DT(388)) to human interleukin-3 (DT(388)IL3) kills malignant progenitors from some patients with acute myeloid leukemia (AML) while sparing normal progenitors. This study evaluated two variants of DT(388)IL3 with increased affinity for the IL-3 receptor (IL-3R) for their cytotoxicity to AML progenitors and determined the ability of quantitative reverse transcription-PCR assessment of expression of the IL-3R subunits to predict the effectiveness of wild-type DT(388)IL3 and its variants. Both the IL-3 deletion variant (Delta125-133) and the amino acid substitution variant (K116W) showed enhanced toxicity against AML colony-forming cells (AML-CFC; but not normal CFC) compared with wild-type DT(388)IL3 with the K116W variant achieving >90% AML-CFC kill with 17 of 23 patient samples. This variant was also more effective against AML cells engrafting in nonobese diabetic severe combined immunodeficient mice. There was a significant correlation between the expression of the alpha and, particularly, the common beta subunit of the IL-3R on AML blasts detected by quantitative reverse transcription-PCR and AML-CFC kill. Thus, the combined use of IL-3R expression to select patients most likely to respond to DT(388)IL3 and the improved cytotoxicity of the K116W DT(388)IL3 variant against leukemic progenitors may enhance the clinical usefulness of these fusion proteins.