Four new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor alpha) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K(D)) 4.5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity Fcgamma-receptor III (CD16), created a bispecific single chain Fv (bsscFv). This bsscFv [123 x ds16] mediated potent lysis of AML-derived MOLM-13, THP-1 and SKNO-1 cells in antibody-dependent cellular cytotoxicity (ADCC) reactions at picomolar concentrations. The recruitment of CD16-positive effector cells for the lysis of AML cells via CD123 represents a novel combination with attractive prospects for future clinical testing.