Recently, we have identified a novel 1.8 kb human Notch4/Int3 RNA species (designated h-Int3sh). The h-Int3sh RNA encodes a protein that is missing the CBF1-binding region (RAM23) of the Notch 4/Int3 intracellular domain (ICD). Expression of h-Int3sh in the MCF10A ‘normal’human mammary epithelial cell line has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth in soft agar. To study the consequences of h-Int3sh expression in vivo on mammary gland development and tumorigenesis, three transgenic mouse lines were established, in which the transgene is the Whey acidic protein (WAP) promoter linked to h-Int3sh. Expression of WAP-Int3sh was detectable in the mammary gland at day 15 of pregnancy in each transgenic line. Mammary gland development in all founder lines is normal and the females can lactate. WAP-h-Int3sh females from each of the founder lines develop mammary tumors, but with a long latency (average age of 18 months). Tumor development was associated with activation of Notch pathway, as evidenced by upregulation of Hes-1. The long latency of mammary tumors in WAP-h-Int3sh mice could be due in part to the subcellular localization of h-Int3sh. Immunofluorescence analysis of transfected COS-1 cells showed that h-Int3sh is localized in the cytoplasm and nucleus, while Int3-ICD is detected only in the nucleus. We speculate that the Notch4/Int3 ICD-induced block to mammary gland development and tumorigenesis are consequences of an increasing gradient of CBF1-dependent Notch4/Int3 signaling.