A transfer system that enabled the efficient introduction of transgenes into neurones and the quantitative control of the expressed transgene would greatly facilitate studies into neuronal gene function. To develop such a system we incorporated the tetracycline (Tet)‐responsive On/Off regulatory elements into type‐5 adenoviral (Ad) vectors. Regulation of transgene expression following transfection was measured by placing the enhanced green fluorescent protein (EGFP) gene upstream of the Tet regulatory element. The results showed that cultures of primary hippocampal cells could be transfected with very high efficiency (<70%) by the AdTet‐On and AdTet‐Off systems. Following transfection with the AdTet‐On system no EGFP‐fluorescent cells could be detected until doxycycline was added. The AdTet‐Off system showed the reverse transcriptional regulation, in that the addition of Tet caused EGFP fluorescence to be abolished.