The ectopic expression of antibody fragments within mammalian cells is a challenging approach for interfering with or even blocking the biological function of the intracellular target. For this purpose, single-chain Fv (scFv) fragments are generally preferred. Here, by transfecting several mammalian cell lines, we compared the intracellular behavior of two scFvs (13R4 and 1F4) that strongly differ in their requirement of disulphide bonding for the formation of active molecules in bacteria. The scFv 13R4, which is correctly folded in the bacterial cytoplasm, was solubly expressed in all cell lines tested and was distributed in their cytoplasm and nucleus, as well. In addition, by appending to the 13R4 molecules the SV40 T-antigen nuclear localisation signal (NLS) tag, cytoplasmic-coexpressed antigen was efficiently retargeted to the nucleus. Compared to the scFv 13R4, the scFv 1F4, which needs to be secreted in bacteria for activity, accumulated, even with the NLS tag, as insoluble aggregates within the cytoplasm of the transfected cells, thereby severely disturbing fundamental functions of cell physiology. Furthermore, by replacing the NLS tag with a leucine-rich nuclear export signal (NES), the scFv 13R4 was exclusively located in the cytoplasm, whereas the similarly modified scFv 1F4 still promoted cell death. Coexpression of NES-tagged 13R4 fragments with nuclear antigen promoted its efficient retargeting to the cytoplasm. This dominant effect of the NES tag was also observed after exchange of the nuclear signals between the scFv 13R4 and its antigen. Taken together, the results indicate that scFvs that are active in the cytoplasm of bacteria may behave similarly in mammalian cells and that the requirement of their conserved disulphide bridges for activity is a limiting factor for mediating the nuclear import/export of target in a mammalian cell context. The described shuttling effect of antigen conferred by a soluble scFv may represent the basis of a reliable in vivo assay of effective protein- protein interactions.