Following intraduodenal priming of mice with reovirus, precursor cytotoxic T lymphocytes (pCTL) rapidly appear in intraepithelial lymphocytes (IEL) and Peyer's patches. These cells express CTL activity after secondary in vitro stimulation with reovirus-infected cells. Adoptive transfer of Peyer's patch lymphocytes from normal BALB/c mice into reovirus-infected CB.17 severe combined immunodeficiency mice results in the infection-dependent appearance of large numbers of both CD8+Thy-1+ and CD8-Thy-1+, IEL that express the alpha/beta T cell receptor (TcR). Phenotypic and functional characterization of IEL derived from conventionally reared, reovirus-infected mice also points to extensive similarities in the pCTL derived from Peyer's patches and IEL. As in the Peyer's patches, pCTL are persistent in the IEL compartment for up to 4 weeks after infection. A large percentage of IEL that are recovered from reovirus-primed mice after in vitro culture are CD8+Thy-1+ cells that express alpha/beta TcR. Furthermore, depletion experiments demonstrate that the CD8+Thy-1+ population mediates the virus-specific CTL activity. Using limiting dilution analyses, it was estimated that 7 days after intraduodenal infection the average frequency of virus-specific pCTL was 197/10(6) CD8+Thy-1+ IEL and 190/10(6) CD8+Thy-1+ Peyer's patch lymphocytes. Taken together, these observations provide evidence that specific cellular immunity to reovirus in IEL is mediated at least in part, by conventional cytotoxic T lymphocytes and that these cells are functionally and phenotypically similar to the pCTL derived from the Peyer's patches.