A new method for investigating cortical microcircuitry uses adenovirus to introduce enhanced green fluorescent protein (EGFP) as a reporter gene to small groups of neurons. Adenovirus in solution containing 600 mM NaCl was injected into the cerebral cortex in anesthetized rats and monkeys, resulting in many EGFP-positive neurons in the interconnected distant brain regions as well as at the injection site. This result suggests that adenovirus with high NaCl concentration will be a kind of retrograde tracer. Thus, I have succeeded in finding a condition of adenovirus injection to retrogradely label cortical neurons to the full extent of their dendritic configurations. This system can be used to study the microcircuitry of central nervous system, and specific mammalian gene function within identified circuits in vivo using RNA interference and/or gene overexpression.