Studies of mammary epithelial cells (MECs) cultured with reconstituted basement membrane proteins derived from EHS tumors have contributed greatly to the understanding of both normal physiology and transformation. Only when plated on such biologically relevant substratum are MECs able to form morphologically correct, differentiated structures, highlighting a critical role for extracellular matrix (ECM) proteins in MEC organization and function. Here, we describe methods modified from the original EHS matrix protocol for isolating tissue-specific ECM from rat mammary glands, and for subsequent use in short-term 3D cell culture models designed to assess acute cell-ECM interactions. Using this protocol, the final matrix is enriched up to 58-fold for ECM proteins such as fibronectin and laminin, while cellular proteins such as GAPDH are reduced 98-fold. We have previously shown that MECs plated in mammary-specific ECM form more elaborate duct-like and alveolar-like structures compared to MECs plated in Matrigel™, demonstrating the biological relevance of tissue-specific ECM. Use of mammary-specific ECM in 3D cell culture models will further our ability to study the intricate interplay between a cell and its microenvironment, and permit identification of modifying factors.