Gemtuzumab ozogamicin (GO) is an anti-CD33 antibody conjugated with calicheamicin (a cytotoxic antibiotic) that is used for the treatment of acute myeloid leukemia (AML). The relationship between the CD33 expression in leukemic cells and response to GO treatment has been controversial. We studied CD33 transcript and protein expression as well as polymorphisms in the CD33 gene in 22 uniformly treated pediatric AML patients and correlated the results with minimal residual disease (MRD) findings before and after GO. We found that a nonsynonymous coding change (Ala14Val) in CD33 was significantly associated with response to GO (P = .02) whereas CD33 transcript and protein expression were not (P > .2). The results suggest a novel mechanism of resistance to GO, one that may extend to other immunotoxins.