Analysis of genes and proteins involved in lipid biosynthesis in mammary epithelial cells (MECs) is complicated by the presence of adipose tissue in the mammary gland, which may be predominant in whole tissue lysates depending upon developmental stage. We have developed a method based on protocols used to establish primary mammary epithelial cell cultures that allows for analysis of MECs depleted of adipose. Adipose depletion yields enriched MECs that are suitable for gene expression profiling and protein analysis from a single mouse. Additionally, the phosphorylation of proteins is maintained, allowing investigation of signal transduction events. Application of this method to the analysis of MECs from genetically modified mice will aid in the identification of factors controlling tissue-specific events in the mammary gland. In contrast to other methods such as laser capture microdissection, the MEC enrichment method described here is performed using standard lab supplies, equipment, and techniques.