Normal developmental events such as puberty, pregnancy, and parity influence the susceptibility of the mammary gland to tumorigenesis in both humans and rodent model systems. Unfortunately, constitutive transgenic mouse models that rely on mammary‐specific promoters to control transgene expression have limited utility for studying the effect of developmental events on breast cancer risk since the hormonal signals governing these events also markedly influence transgene expression levels. A novel transgenic mouse system is described that uses the MMTV‐LTR to drive expression of the reverse tetracycline‐dependent transactivator rtTA. Transgenic mice expressing rtTA in the mammary epithelium were crossed with reporter lines bearing tet operator‐controlled transgenes. We tested the ability to spatially, temporally, and quantitatively control reporter gene expression after administration of doxycycline to bitransgenic mice. Transgene expression using this system can be rapidly induced and deinduced, is highly mammary specific, can be reproducibly titrated over a wide range of expression levels, and is essentially undetectable in the uninduced state. Homogeneous transgene expression throughout the mammary epithelium can be achieved. This system permits transgene expression to be restricted to any desired stage of postnatal mammary gland development. We have developed a mammary‐specific, doxycycline‐inducible transgenic mouse model for studying the effect of mammary gland development on transgene‐mediated phenotypes. Unlike other mammary‐specific, transgenic systems that have been described, this system combines spatially homogeneous transgene expression in the mammary epithelium during puberty, pregnancy, lactation, and involution with the use of an orally administered, inexpensive, and widely available inducing agent. This system offers new opportunities for the transgenic analysis of mammary gland biology in vivo.—Gunther, E. J., Belka, G. K., Wertheim, G. B. W., Wang, J., Hartman, J. L., Boxer, R. B., Chodosh, L. A. A novel doxycycline‐inducible system for the transgenic analysis of mammary gland biology. FASEB J. 16, 283–292 (2002)