Orthotopic and ectopic organ environments differentially influence tumor growth, metastasis, and sensitivity to therapy. In this study we present a novel rodent mammary window of orthotopic breast cancer, which is amenable to study of microvascular function and angiogenesis in this orthotopic site. The skin around the nipple of selected mammary glands of female Fischer 344 rats was removed and the nipple was cut at its base. R3230Ac tumor fragments or cells in Gelfoam were aseptically implanted into the nipple sinus. An acrylic disk was placed on top of the implant and was sutured in place. Histology showed that tumors were well established within 5 days. Similar techniques were also applied to BALB/c mice transplanted with 4T1 murine mammary carcinoma cells. With GFP-expressing tumor cells and serial observations, we demonstrated unique patterns of tumor cell proliferation and vascularization in both tumor models. The images obtained were comparable to those from the dorsal skinfold window chambers. This model will allow for study of tumor microcirculatory function, angiogenesis, tumor cell-host interactions, and evaluation of effects of various treatments.