Nonviral, plasmid-based gene transfer into somatic tissues offers the prospect of various simple and safe therapeutic possibilities as well as applications in fundamental research. Although cationic lipids display efficient transfection activities in many in vitro systems, only low success rates using these vectors in vivo have been reported. We succeeded in defining conditions providing high levels of in vivo transfection in the brains of newborn mice. Our hypothesis was that conditions favorable for in vitro transfection (highly positively charged particles) were unlikely to be appropriate for in vivo conditions. When using the cationic lipid dioctadecylamido glycylspermine (Transfectam, DOGS) with a cytomegalovirus (CMV)-luciferase reporter gene, the best levels of transfection were obtained when using a low ratio of positive charges (supplied by the DOGS) to negative charges (carried by the DNA). Moreover, addition of the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) significantly enhanced transfection. Expression of the transgene diminished over time, independently of lipopolysaccharide content of the plasmid preparation used. This suggests that either a mitotic population of cells was preferentially transfected, or that promoter silencing was occurring. Histological examination of the spatial distribution of a beta-galactosidase-expressing transgene showed numerous groups of transfected cells both within the striatal parenchyma and in the paraventricular area. Thus, DNA-lipid complexes bearing overall charges close to neutrality open promising possibilities for modulating gene expression in the developing central nervous system and for therapy in the brain.