The tetracycline-regulatable system (TRS) has become a widely adopted tool for modification of gene expression and analysis of gene function in mammalian cells, plants and transgenic animals. We have studied the potential application of the TRS in gene therapy, using a single vector containing both the tetracycline-controlled transactivator (tTA) and the tTA-responsive promoter (tRP) transcribing mouse GM-CSF. Stable 293 cells established using this vector were used to study the kinetics of the TRS in response to various tetracycline analogues. Dose–response studies show that doxycycline is the most potentanalogue in abolishing tTA activity. Kinetic studies indicate that, at 1000 ng/ml, all the analogues have similar efficiencies in down-regulating the system in a given time. In contrast, following the removal of the analogues, there is a temporal, dose-dependent delay in resumption of the tRP activity. The time taken for resumption of near-optimal tRP activity is approximately 48 h for tetracycline, 144 h for anhydrotetracycline, 192 h for minocycline and 216 h for doxycycline when cells were pretreated with 1000 ng/ml of these antibiotics. This property of the analogues can be employed in planning a desired course of transgene regulation.