SUMMARY: MDCK cells dissociated from monolayer culture were either dispersed within medium-hydrated collagen gel or seeded atop a collagen substrate which was immediately overlaid with collagen gel. Individual cells exhibited clonal growth in three dimensions to form spherical cysts in which a simple epithelium surrounded a fluid-filled lumen. The ceils of MDCK cysts were polarized with apical surface bordering the lumen. MDCK cysts increased in diameter with continued culture. Maximum cyst size was dependent on seeding density and was influenced by medium composition. MDCK cysts could be isolated from the collagen substrate by digestion with collagenase. Also, collagen gel could be dissected from the cyst wall to give unrestricted access to regions of the basolateral cell surface. This novel method of renal cell culture provides a study system to model the influence of the extracellular matrix on kidney epithelial cell structure and function. It also offers an in vitro model of general application to the study of epithelial cyst formation and growth.