We have developed an efficient and cell-specific nonviral gene delivery system using monoclonal antibodies (mAb) coupled with branched 25-kDa polyethyleneimine (PEI). The system was evaluated for two model antibodies with different well-characterized antigen specificities: the mouse anti-human IgG1 mAb AS02 recognizing human CD90 (hThy-1) which is expressed on human fibroblasts, and the humanized anti-Her-2/neu mAb Trastuzumab recently introduced for the treatment of Her-2/neu-positive breast cancer. Efficacy and selectivity of gene delivery were evaluated for covalent mAb-PEI conjugates coupled with N-succinimidyl-3-(2-pyridyldithio)proprionate (SPDP) or N-succinimidyl-4-(maleimidomethyl)-cyclohexancarboxylate (SMCC), or, as a newly introduced coupling reagent, noncovalent complexes of mAb with 3-(2-(2-(vinylsulfonyl)ethylthio)ethyl)quinazoline-2,4(1H,3H)-dione (IBFB 110001) coupled to PEI. An enhanced green fluorescent protein (EGFP)-expressing reporter plasmid was used to monitor transfection efficiencies of cell lines expressing different levels of hCD90 or Her-2/neu. While mAb-PEI conjugates coupled with SPDP resulted in antigen-nonspecific EGFP expression, conjugates coupled with SMCC or IBFB 110001 enabled antigen-specific gene delivery. Thus, Her-2/neu-PEI conjugates prepared with IBFB 110001 allowed to transfect 23+/-2% of Her-2/neu-positive SKOV-3 versus 0.5+/-2% of Her-2/neu-negative MB-468 cells. Proof of principle for specific antibody-mediated gene transfer was demonstrated by saturating the Her-2/neu receptor with free anti-Her-2/neu, thereby blocking subsequent transfection with anti-Her-2/neu-PEI/DNA complexes.