The aim of this study was to establish a long-term culture system for rat colon epithelial cells. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salt]–containing buffer, taken up in conditioned medium from the normal rat kidney fibroblast cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C–treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin 19 and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating factors on growth and gene expression of normal colon epithelial cells in vitro.