Single-chain antibody variable fragment (scFv) proteins consist of an antibody heavy chain variable sequence joined via a flexible linker to a light chain variable sequence. Prior work has shown that ScFv 18-2 binds the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and sensitizes cancer cells to radiation following nuclear microinjection. A potential clinical delivery strategy is based on modification of the scFv so that it can be taken up into cells and imported to the nucleus. This will require development of an expression system for a nuclear localization signal (NLS)-tagged scFv derivative. We found, however, that addition of the highly basic NLS severely compromised expression in the host-vector system used for the parental scFv. After testing a variety of host strains, fusion partners, and NLS sequences and placements, successful expression was obtained with a construct containing a stabilizing N-terminal maltose binding protein tag and a single, optimized, C-terminal NLS moiety. Amylose affinity-purified ScFv 18-2 NLS protein was stable to storage at 4 degrees C in the presence of glycerol or trehalose, bound selectively to an epitope peptide, and was cleavable at an engineered Factor Xa protease site. Following lipid-mediated uptake into cultured cells, NLS-tagged ScFv 18-2, unlike the parental ScFv 18-2, localized predominantly in the cell nucleus.