Under physiological salt concentration, plasmid DNA complexed with transferrin-conjugated or unmodified polyethylenimine (PEI, 800 kDa) forms huge (up to > 1000 nm) aggregates, unless the individual components are mixed at a highly positive nitrogen/phosphate (N/P) charge ratio. At low ionic strengths, however, small particles with an average size of 40 nm are formed over a broad range of N/P ratios. Interestingly, in transfection experiments these small particles result in a 10-fold (B16F10 cells) to more than 100-fold (Neuro2A cells, K562 cells) reduced luciferase gene expression efficiency in comparison to the large complexes formed in physiological salt solutions. Limited transport of the small particles to the cell surfaces is one possible reason for this effect. Application of the small particles in more concentrated form and over extended periods of time improves transfection activity. Reduced intracellular release may be another explanation for the decreased transfection efficiency; incubation with chloroquine or incorporation of the endosomolytic peptide INF5 into the small complexes enhances gene expression approximately 10-fold. Analysis of gene expression at the cellular level using a green fluorescence protein reporter gene and flow cytometry revealed that the differences in overall gene expression largely result from different intensities per expressing cell, while the difference in the percentage of expressing cells is less substantial.