The susceptibility of the mammary epithelium to neoplastic transformation is linked to the exposure to estrogen during the estrous cycle. The effects of the estrous cycle on the mouse mammary gland have been investigated by analyzing 3H-thymidine incorporation, milk protein gene expression and DNA fragmentation. We found that the mammary epithelium undergoes limited proliferation, differentiation and apoptosis in a cycle-dependent manner. The estrous-responsive regulators of the mammary epithelium are unknown; however, considering the integral role of protein tyrosine kinases (PTKs) in the control of normal and malignant development, members of this family of enzymes are likely candidates for such regulatory molecules. Using a RT-PCR-based cloning strategy, we have undertaken a survey of PTKs expressed in the mammary gland at defined stages of development, with special emphasis on the estrous cycle. We identified 21 known and 4 novel PTKs. Their expression was analyzed throughout mammary gland development and in mammary neoplasias using a transgenic mouse model for invasive and non-invasive carcinogenesis. Most of the identified PTKs showed highest expression during the estrous cycle and were down-regulated during pregnancy and lactation. Deregulated expression was rarely observed in the non-invasive mammary tumors. In contrast, 10 of 19 PTKs expressed during the estrous cycle were also over-expressed in the invasive carcinomas, mostly involving members of the receptor family of PTKs.