This protocol describes a method for directing the expression of genes of interest into postmitotic neocortical neurons in vivo. Microinjection of a DNA plasmid-amphiphilic molecule mix into the neocortex followed by delivery of an ad hoc electric pulse protocol during the first few days of life in mice allows rapid, focal and efficient expression of genes in postmitotic neurons. Compared with other gene delivery techniques such as in utero electroporation and viral infection, this method allows rapid (12 h), focal (50-200 μm), mosaic-like (50 to several hundred neurons) targeting of postmitotic neurons within existing circuits. This 'iontoporation' protocol, which can be completed within ∼20 min per mouse, allows straightforward assessment of genetic constructs in postmitotic cortical neurons and subsequent genetic, histological and physiological investigations of gene function.