Epithelial cells grown in three-dimensional (3-D) cultures of extracellular matrix differentiate into a multicellular structure of polarized cells. This process shares many characteristics with the physiological development of an epithelial tissue and the formation of polarity in epithelial cells. Imaging 3-D cultures of polarized epithelial cells is therefore a powerful tool to study epithelial architecture and morphogenesis under close-to-physiological conditions. The new generation of confocal microscopes allows live-cell imaging of fluorescently tagged molecules in these cultures. This opens up new opportunities for studying how molecules behave and are distinguished asymmetrically within a 3-D setting. This unit discusses technical aspects for culturing and imaging MDCK 3-D culture for both fixed 3-D cultures and live-cell imaging.