We have previously documented the cell-type-specific and hormone-dependent expression of the EphB4 receptor in the mouse mammary gland. To investigate its role in the biology of the mammary gland, we have established transgenic mice bearing the EphB4 receptor under the control of the MMTV-LTR promoter, which represents the first transgenic mouse model to investigate the effect(s) of unscheduled expression of EphB4 in adult organisms. Transgene expression in the mammary epithelium was induced at puberty, increased during pregnancy, culminated at early lactation and persisted until day three of post-lactational involution. In contrast, expression of the endogenous EphB4 gene is downregulated during pregnancy, is essentially absent during lactation and is re-induced after day three of post-lactational involution. The unscheduled expression of EphB4 led to a delayed development of the mammary epithelium at puberty and during pregnancy. During pregnancy, less lobules were formed, these however exhibited more numerous but smaller alveolar units. Transgenic mammary glands were characterized by a fragile, irregular morphology at lactation; however, sufficient functionality was maintained to nourish the young. Transgenic mammary glands exhibited untimely epithelial apoptotic cell death during pregnancy and abnormal epithelial DNA synthesis at early post-lactational involution, indicating a disturbed response to proliferative/apoptotic signals. Mammary tumours were not observed in the EphB4 transgenic animals; however, in double transgenic animals expressing both EphB4 and the neuT genes, tumour appearance was significantly accelerated and, in contrast to neuT-only animals, metastases were observed in the lung. These results implicate EphB4 in the regulation of tissue architecture, cellular growth response and establishment of the invasive phenotype in the adult mammary gland.