Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ERalpha) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ERalpha expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ERalpha placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mERa mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mERalpha mice were produced. All three lines demonstrated dominant gain of ERalpha shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ERalpha was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ERalpha dominant gain in targeted tissues at specific times during development. In this study dominant gain of ERalpha was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ERalpha on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ERalpha action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ERalpha can rescue identified phenotypes in gene knockout mice.