We have developed an advanced method for conditional gene expression in mice that integrates the Cre-mediated and tetracycline-dependent expression systems. An rtTA gene, preceded by a loxP-flanked STOP sequence, was inserted into the ROSA26 locus to create a R26STOPrtTA mouse strain. When the STOP sequence is excised by Cre-mediated recombination, the rtTA is expressed in the Cre-expressing cells and all of their derivatives. Therefore, cell type-, tissue-, or lineage-specific expression of rtTA is achieved by the use of an appropriate Cre transgenic strain. In mice also carrying a target gene under the control of the tetracycline response element, inducible expression of the target gene is temporally regulated by administration of doxycycline. Our results demonstrate that this universal system is uniquely suited for spatiotemporal and lineage-specific gene expression in an inducible fashion. Gene expression can be manipulated in specific cell types and lineages with a flexibility that is difficult to achieve with conventional methods.