Mammalian cells in culture can be modified by gene transfer and these procedures are routinely used in experimental biology. Yet, efficient approaches to modify certain complex cell populations, stem cells or cells organized within a tissue are still lacking. The hurdles to gene transfer can be listed by describing the pathway of a nucleic acid molecule, from the external medium towards the cell nucleus where its encoded information will be expressed. The requirements include the necessity to compact the size of the macromolecule, to overcome electrostatic repulsion, to cross a series of membranes and to establish itself permanently. Viruses have evolved to achieve these goals and understanding their strategies for cell invasion allows to design vectors for gene transfer. Chemicals or biochemicals able to bind DNA, as well as physical methods inducing changes in the structure of membranes can also be useful for gene transfer. The outcome of gene transfer technologies and their improvement pave the way to inovative medical applications and provide powerful tools for exploring the living world.