The transgenic mouse strains surfactant protein C-reverse tetracycline transactivator (SP-C-rtTA), club cell secretory protein (CCSP)-rtTA, and tetracycline operator (TetO)-Cre have been invaluable for spatiotemporally regulating gene deletion in the pulmonary epithelium. In this study, we measured the efficiency and specificity of gene deletion that can be achieved in these mice using the Rosa26-eYFP reporter. Triple-transgenic mice (tTg or rtTA/TetO-Cre/Rosa-eYFP) were bred and treated with various doxycycline (dox) regimens to induce gene deletion, which was then quantified in various cell populations by flow cytometry. In these crosses, we found that the TetO-Cre transgene must be transmitted through the female parent to avoid germline gene deletion. With dox exposure during lung development, SP-C-tTg mice deleted in ˜65-75% of alveolar epithelial type II (ATII) cells, but in only ˜45-50% of the integrin β4+ population, which consisted of club cells and distal lung progenitor cells. In contrast, CCSP-tTg mice deleted in ˜50% of ATII cells and ˜80% of integrin β4+ cells. Upon dox treatment of adults, deletion in ATII cells and integrin β4+ cells in SP-C-tTg mice dropped significantly to ˜20% and ˜6%, respectively, whereas CCSP-tTg mice deleted in ˜57% of ATII and ˜40% of integrin β4+ cells. Interestingly, untreated CCSP-tTg mice also deleted in ˜40% of integrin β4+ cells, indicating significant leakiness of CCSP-tTg in β4+ cells. In all mouse groups, minimal deletion occurred in mouse tracheal epithelial cells or in mesenchymal or hematopoietic cells. These data provide the first quantitative, side-by-side comparison of the deletion efficiency for these widely used transgenic mouse strains.