Previously, we reported the evaluation of several polyplex-based gene delivery systems with respect to their effectiveness, toxicity, and cell type dependence in vitro. One system, P123-g-PEI(2K), a cationic graft block copolymer, is of particular interest as it has been demonstrated to successfully deliver genetic material to murine liver following systemic delivery [Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Guerin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of Polyether-Polyethyleneimine Graft Copolymers as Gene Transfer Agents. Gene Ther. 7, 126-138 (1)]. The P123-g-PEI(2K) system requires nonmodified Pluronic P123 as an excipient to stabilize the dispersion. The purpose of the current work was to more closely characterize this system, to assess the role of each component of the system to the overall transfection process. We evaluated particle size, stability, and resistance to nuclease degradation. In addition, cellular uptake and localization of plasmid, as well as transgene expression, were evaluated following in vitro transfection of prostate cancer cells (PC-3) with various individual components of the system. Nonmodified Pluronic alone did not significantly enhance DNA uptake, transgene expression, or DNase protection. Therefore, we conclude that nonmodified Pluronic acted primarily by optimizing the size of the polyplex. Furthermore, though this system displays several characteristics thought desirable of a nonviral gene delivery system, these studies did discriminate a potential limitation of this system for in vivo applications, namely, the insufficient level of protection of plasmid DNA from nuclease degradation. This may limit the effective dose delivered, as well as limiting the effective circulation time. These studies provide vital information that will guide modification of this system to enhance the current in vivo profile.