In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variable transfection efficiency depending on the concentration of oligonucleotide used. A significant population of cells, especially at lower doses of oligonucleotide and cationic lipid, were untransfected. We investigated lipid/oligonucleotide ratios, different lipid preparations, and different cell types and found that these variables did not alter the percentage of cells transfected at these lower doses of oligonucleotide. However, when lipid-oligonucleotide complexes were formed at the high dose and then diluted into a solution of lipid or a complex of lipid and unlabeled, negative control oligonucleotide, a constant percentage of cells was transfected. Under these conditions, mRNA target reduction dose-response curves were also shifted to lower doses. We hypothesize that poor transfection observed at a low concentration of lipid-oligonucleotide complex when diluted in medium is due to loss of active complexes, either by adsorption to the substrate or by changes in physical characteristics of complexes. By maintaining a constant lipid concentration, more consistent transfection was achieved.