Techniques for isolating the desired cell populations from complex tissues are essential for characterizing cells through mRNA analysis. We established a procedure for isolating pure mucosal epithelium from the human alimentary tract. To do this, we made rotating hooks that hold mucosal strips and detach the epithelial sheets from the irregular mucosae surface in medium containing EDTA. An additional step using a cell strainer was required to reduce contamination by lymphoid cells. Sheets of epithelial cells were detached successfully from mucosal samples derived from five different parts of the human alimentary tract. Contamination by lymphoid cells or fibroblasts was monitored by competitive RT-PCR and was no more than 0.5% of the total cells. Total RNA yields were 12.5-17 micrograms for each separation, and the integrity of the RNA was as good as that of RNAs extracted from mucosa immediately after resection. In conclusion, our method permits isolation of RNAs from a pure population of epithelial cells that can be used for mRNA-based gene expression analyses.