The use of a nonviral magnetic vector, comprised of magnetic iron oxide nanoparticles (MNP), polyethylenimine (PEI), and plasmid DNA, for transfection of BHK21 cells under a magnetic field is presented. Four different vector configurations were studied by systematically varying the mixing order of MNP, PEI, and DNA. The assembly of the vector has significant effects on its vector size, surface charge, cellular uptake, and level of gene expression. Mixing MNP with PEI first improved MNP stability, giving a narrow aggregate size distribution and positive surface charge at physiological pH, which in turn facilitated DNA binding onto MNP. The presence of serum in culture media improves vector dispersion and alters the surface charge of all vectors to negative charge, indicating serum protein adsorption. Cellular uptake was greater for larger vectors than the smaller vectors due to enhanced gravitational and magnetic aided sedimentation onto the cells. High MNP uptake by the cells, however, does not inevitably lead to increase gene expression efficiency. It can be shown that besides vector uptake, gene expression is affected by extracellular factors such as premature DNA release from MNP and DNA degradation by serum as well as intracellular factors such as vector lysosomal degradation, inability of DNA to detach from MNP, and cytotoxic effects of MNP at high uptake. Some of these extra- and intracellular properties are shown to be mediated by the presence of PEI.