The aim of this study was to develop a method by which colonic epithelial cells can be isolated from resected mucosa or colonoscopic biopsy specimens and viably maintained in the short term. The principles of the technique are to digest the lamina ropria from the epithelium with Dispase and collagenase, to disrupt the epithelium by trituration, and to purify the epithelial cells by sieving and differential sedimentation. Whole and partial crypts were isolated with consistently high purity of 93.5% ± 1.2% (excluding red cells). Structural integrity was confirmed by light and electron microscopy, exclusion of trypan blue, minimal leakage of lactic dehydrogenase over 5 h (4.1% ± 1.7%), and 51Cr leakage of <2% per hour over 16 h. Functional integrity was supported by continued deoxyribonucleic acid synthesis ([3H]thymidine uptake) over 16 h and the formation of epithelial monolayer cultures on plastic. Thus, this simple method yields a highly enriched cell population that maintains high viability in vitro for at least 16 h. Such cells may be useful for the study of the biology of colonic epithelial cells.