The transfection efficiencies of 25-kDa branched polyethylenimine (B-PEI) and 22-kDa linear PEI (L-PEI) with both DNA and small interfering RNA (siRNA) were compared and correlated with their biophysical properties relating to complex formation, stability, and disassembly. L-PEI-DNA complexes transfected (5.18 × 10(8) relative luminescence units [RLU]/mg) around fivefold better than B-PEI-DNA complexes (0.95 × 10(8) RLU/mg), whereas B-PEI-siRNA complexes gave approximately 60% gene knockdown and L-PEI-siRNA complexes were inactive. Both B-PEI and L-PEI packaged DNA and siRNA to form positively charged nanoparticles; however, L-PEI nanoparticles were less stable than B-PEI nanoparticles, particularly with siRNA. The poor stability of L-PEI-siRNA complexes seemed to be the major factor contributing to an observed lack of cellular uptake and hence poor transfection. The more stable B-PEI-siRNA complexes, however, were bound, internalized, and detectable in the cytoplasm. These results highlight the importance of particle stability for efficient siRNA and plasmid delivery, while retaining the ability to readily dissociate within the cell. FROM THE CLINICAL EDITOR: Comparison of branched versus linear cationic polymers, i.e, polyethylenimine (PEI), were compared for their formation of condensed DNA and SiRNA complexes. Branched complexes were superior for transfection due to improved structural stability, making this PEI approach more likely to succeed as a nanotherapy.