A single chain antibody (scFv) was constructed from a hybridoma expressing the anti-CD34 monoclonal antibody My10. The scFv was expressed in the mouse fibroblast cell line NIH 3T3, and purified from culture supernatant via an epitope tag fused to the C-terminus of the protein. The scFv equilibrium dissociation constant (KD) was determined to be 2.4 X 10(-7) M using a fluorescence based flow cytometry assay involving recognition of the epitope tag, and bound approximately 24-fold less avidly to CD34 expressing KG-1a cells than the native antibody My10. This novel and previously unreported method for determining antibody binding affinity offers several advantages over alternative methods. It is rapid and simple, and unlike methods that directly label the antibody, it involves no covalent modifications of antibody variable domain residues that could potentially interfere with antigen binding. The KD for the anti-CD33 antibody HuG1 (Caron et al. (1992) The biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res. 52, 6761-6767) was determined as well. The close agreement of this value and the previously reported value, determined by a radioligand competition method, validates the use of this assay for antibody affinity determination. We discuss various potential applications for this anti-CD34 scFv.