A tamoxifen-dependent Cre recombinase (Cre-ER^T) was developed for conditional site-specific recombination in mice. This recombinase is activated by tamoxifen but not by estradiol. Transgenic mice expressing Cre-ER^T under a cytomegalovirus promoter were generated, allowing the inducible excision of a chromosomally integrated gene flanked by loxP sites upon tamoxifen administration. This system enables the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting. The study demonstrates that Cre-ER^T can efficiently excise the tkneo gene in F9 cells when treated with tamoxifen, but not with estradiol. In transgenic mice, tamoxifen treatment led to excision of the floxed tkneo marker in various organs, except the thymus. The level of Cre-ER^T mRNA correlated with the efficiency of DNA excision, with the highest excision rates observed in the tail, skin, kidney, and spleen. The system allows for the generation of genetic mosaics, enabling the analysis of gene function in a tissue-specific and time-controlled manner. This approach overcomes limitations of conventional gene targeting, such as the inability to analyze gene function in postnatal development or in tissues where gene inactivation leads to embryonic lethality. The study also highlights the potential of this system for creating somatic mutations in a spatio-temporally controlled manner. The results indicate that Cre-ER^T is a tightly regulated recombinase that can be activated by tamoxifen treatment in mice, with no observed adverse effects. This system represents a significant advancement in the study of mammalian development and physiology.A tamoxifen-dependent Cre recombinase (Cre-ER^T) was developed for conditional site-specific recombination in mice. This recombinase is activated by tamoxifen but not by estradiol. Transgenic mice expressing Cre-ER^T under a cytomegalovirus promoter were generated, allowing the inducible excision of a chromosomally integrated gene flanked by loxP sites upon tamoxifen administration. This system enables the analysis of knockout phenotypes that cannot be addressed by conventional gene targeting. The study demonstrates that Cre-ER^T can efficiently excise the tkneo gene in F9 cells when treated with tamoxifen, but not with estradiol. In transgenic mice, tamoxifen treatment led to excision of the floxed tkneo marker in various organs, except the thymus. The level of Cre-ER^T mRNA correlated with the efficiency of DNA excision, with the highest excision rates observed in the tail, skin, kidney, and spleen. The system allows for the generation of genetic mosaics, enabling the analysis of gene function in a tissue-specific and time-controlled manner. This approach overcomes limitations of conventional gene targeting, such as the inability to analyze gene function in postnatal development or in tissues where gene inactivation leads to embryonic lethality. The study also highlights the potential of this system for creating somatic mutations in a spatio-temporally controlled manner. The results indicate that Cre-ER^T is a tightly regulated recombinase that can be activated by tamoxifen treatment in mice, with no observed adverse effects. This system represents a significant advancement in the study of mammalian development and physiology.