Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (V(H) and V(L)) domains of two different specificities connected by three linkers. When assembled in the order V(H)(A)-linker(1)-V(L)(B)-linker(2)-V(H)(B)-linker(3)-V(L)(A), the single-chain molecule either folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody, or forms a homodimer that is twice as large, a so-called tandem diabody. The formation of the tandem diabody is determined by the association of complementary V(H) and V(L) domains located on different polypeptide chains, and depends on the length and probably the amino acid composition of the three linkers joining the variable domains. We generated a number of single-chain constructs using four V(H) and V(L) domains specific either for human CD3, a component of T-cell receptor (TCR) complex, or for CD19, a human B-cell antigen, separated by different rationally designed peptide linkers of 6-27 amino acid residues. The generated bispecific constructs were expressed in bacterial periplasm and their molecular forms, antigen-binding properties, stability, and T-cell proliferative and anti-tumor activities were compared. Using peripheral blood mononuclear cell cultures from patients suffering from B-cell chronic lymphocytic leukemia, we demonstrated that the tandab-mediated activation of autologous T cells and depletion of malignant cells correlates with the stability of the recombinant molecule and with the distance between the CD19 and CD3 binding sites.