Gemtuzumab ozogamicin (GO; Mylotarg), a novel immunoconjugate used for treatment of acute myeloid leukemia (AML), contains the humanized anti-CD33 antibody (hP67.6) as a carrier to facilitate cellular uptake of the toxic calicheamicin-gamma(1) derivative. By use of lentivirus-mediated gene transfer to manipulate CD33 expression in myeloid cell lines that normally lack CD33 (murine 32D cells) or have very low levels of CD33 (human OCI-AML3 and KG-1a cells), we here show a quantitative relationship between CD33 expression and GO-induced cytotoxicity. The CD33 cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) control internalization of antibody bound to CD33. Disruption of the ITIMs by introduction of point mutations not only prevented effective internalization of antibody-bound CD33 but also significantly reduced GO-induced cytotoxicity. Together, our data imply a pivotal role of both the number of CD33 molecules expressed on the cell surface and the amount of internalization of CD33 following antibody binding for GO-induced cytotoxicity and suggest novel therapeutic approaches for improvement of clinical outcome of patients treated with GO.