Mechanical, enzymatic and chelating methods for isolating rat intestinal epithelial cells were compared to determine the best technique for obtaining high yields of viable cells. The mechanical techniques resulted in consistently poor viability compared with the other methods. There was excellent agreement between viability determinations made by trypan blue exclusion and by cytofluorochromasia. Incubation with EDTA, citrate and dithiothreitol, combined with mechanical extrusion of dissociated cells provided the highest yields of viable cells in these studies.