Many epithelial tissues develop deep within the animal and are inaccessible to high-resolution optical imaging with visible wavelengths. Protocols for culturing whole epithelial organs have existed since the 1950s, but the use of three-dimensional (3D) organotypic cultures of epithelial fragments has advanced dramatically in recent years. Here we describe the practical details involved in isolating mammary epithelial tissue and culturing organoids embedded within 3D gels. Mammary glands are mechanically disrupted and enzymatically digested, and the epithelial cell fragments are separated from stromal cells by differential centrifugation. The organoids are cultured in BD Matrigel in the absence or presence of growth factor.