Bispecific antibodies (bi-MABs) can be used to target T cells to autologous tumor cells. It has been shown that the activation of resting human T cells requires two independent signals, namely the cross-linking of the T-cell receptor (TCR)-CD3 complex together with the CD28 homodimer. In the present study, we demonstrate the activation of T cells from patients with chronic lymphocytic leukemia (CLL) using bi-MABs against the CD3 and CD19 antigens (CD3 x CD19) in combination with monospecific, bivalent antibodies against the CD28 antigen. Mononuclear cells from patients with CLL were cultured with the bi-MAB CD3 x CD19 and monospecific CD28 antibodies. The CD3 x CD19 bi-MABs were isolated by the hybridoma-hybridoma fusion technique and purified by hydrophobic interaction chromatography. T-Cell activation as demonstrated by increased proliferation, upregulation of T-cell activation markers (CD25, CD38), and cytotoxicity against autologous CLL cells and allogeneic B cells was shown in seven of eight CLL specimens. The stimulation with CD3 x CD19 bi-MABs with CD28 antibodies preferentially induced proliferation of CD4+ T cells. The effective dose of purified antibodies required for optimal T-cell activation was 100 ng/mL in vitro, which suggests that this antibody combination may be useful for immunotherapy of patients with B-CLL.