To examine the involvement of CD8alpha+ intraepithelial lymphocytes (IELs) in chemokine expression by villus epithelial cells, villus and crypt fractions were collected by mechanical isolation using a chelating buffer and specific antibodies in CD8alpha+ cell-depleted rats. A larger population of CD8alpha+ cells was observed by histochemical evaluation in villus epithelia than in crypt epithelia in rat small intestine, and CCL9 and CCL28 expression was higher in the crypt fraction than in the villus fraction. The mRNA expression of CCL28 in villus fractions isolated from rat small intestinal mucosa was significantly reduced compared to that of CCL9, and was accompanied by CD8alpha depletion. Using a combined histochemical and flow cytometric approach, CD8alphaalpha+ cells were detected in the intraepithelial region of the villus epithelium. Thus, CCL28 expression in villus epithelial cells is partially supported by CD8alphaalpha+ cells, and CD8alpha+ IELs are involved in CCL28 expression.