We have previously shown an age-associated increase in unique CD4+CD8+ intestinal intraepithelial lymphocytes (i-IEL) in rats. To elucidate the potential causes of the increase in CD4+CD8+ i-IEL with age, we analyzed the specificity of the CD4+CD8+ i-IEL and influence of intestinal microflora on the increase in this subset in aged rats. The purified CD4+CD8+ i-IEL proliferated in response to DnaK [Escherichia coli (E. coli) HSP70] in the presence of mitomycin-c (MMC)-treated syngeneic spleen cells. The proportion of CD4+CD8+ T cells in whole i-IEL were significantly increased in aged rats fed commercial (CL-2) diet but not in those fed home-made (purified) diet under conventional condition. No CD4+CD8+ i-IEL were detected in aged rats under germfree condition, irrespective of diet feeding. A larger number of Enterobacteriaceae, especially E. coli, were detected in the intestinal contents and feces from aged rats with CD4+CD8+ i-IEL compared with those from aged rats fed without CD4+CD8+ i-IEL. The unique CD4+CD8+ i-IEL population specific for E. coli HSP may be associated with long term exposure to intestinal E. coli in aged rats.