INTRODUCTION Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, all of which must be circumvented before any gene expression can occur (Figure 12.1). First, vectors must be targeted to specific cell types while avoiding many others. Second, before the vector can reach any cell, it must make its way through the extracellular matrix. Third, the vector must enter the cell by breaking through the plasma membrane/endosome barrier. Finally, once in the cytoplasm of non-dividing cells, the DNA is confronted by the nuclear membrane, which it must traverse to enter the nucleus. That the DNA must enter the nucleus is self-evident: without localization to the nucleus, no transcription, replication, integration, or maintenance can take place. However, given the importance of the nuclear entry step, there has been surprisingly little attention directed toward either discovering or exploiting the mechanisms used by the cell to direct DNA to the nucleus. In this chapter, I will focus on the nuclear import step of gene delivery, which will hopefully lead to an increased appreciation for this barrier.