With the demonstration of improved survival of some acute myeloid leukemia (AML) patients with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), CD33 has been validated as a target for antigen-specific immunotherapy. Since previous studies identified a CD33 splice variant missing exon 2 (CD33∆ E2) and, consequently, the immune-dominant membrane-distal V-set domain, we investigated the expression and functional characteristics of CD33 transcript variants in AML. In primary AML specimens, we not only found full-length CD33 (CD33 FL) and CD33∆ E2 but also corresponding variants containing an alternate exon 7 predicted to encode a CD33 protein lacking most of the intracellular domain (CD33 E7a and, not previously described, CD33∆ E2, E7a) in almost all cases. In acute leukemia cell sublines engineered to express individual CD33 splice variants, all splice variants had endocytic properties. CD33 FL and CD33 E7a mediated similar degrees of GO cytotoxicity, whereas CD33∆ E2 and CD33∆ E2, E7a could not serve as target for GO. Co-expression of CD33∆ E2 did not interfere with CD33 FL endocytosis and did not impact CD33 FL-mediated GO cytotoxicity. Together, our findings document a greater-than-previously thought complexity of CD33 expression in human AML. They identify CD33 variants that lack exon 2 and are not recognized by current CD33-directed therapeutics as potential target for future unconjugated or conjugated antibodies.