The functional and phenotypic heterogeneity of the rat peripheral T lymphocyte antigen RT6 has been examined. The in vivo popliteal graft-vs-host reaction (GvHR), in vitro MLR, and the generation and effector populations of cytotoxic T lymphocyte were used to examine the response of RT6-positive (RT6+) or -negative (RT6-) subsets of CD4 and CD8 T cells to alloantigen. T lymphocytes with the CD4+RT6+ surface phenotype are necessary and sufficient for inducing a strong GvHR. T lymphocytes with the CD4+RT6-, CD8+RT6+, and CD8+RT6- surface phenotypes do not contribute or induce a strong GvHR. Both the CD4+RT6+ and the CD4+RT6- T cells proliferate in the MLR assay. CTL precursors are a mixture of CD8+RT6+ and CD8+RT6- phenotypes, but only cells bearing the RT6- phenotype are potent CTL effector cells. Data presented in this paper also demonstrate that the RT6.1 alloantigen, present on the majority of rat T cells, modulates on cortisone-resistant thymocytes (CRT) and peripheral T cells. Although freshly isolated CRTs do not express the RT6.1 epitope, RT6+ cells develop when CRTs are placed into culture. Peripheral T cells that are RT6- will also become RT6+ in culture. Stimulation of T cell cultures with a mitogen causes the loss of the RT6.1 antigen, as detected by the DS4.23 mAb. The effect is more pronounced in cultures of CRTs than peripheral T cells. Following a return to a nonactivated state, the T cells reexpress RT6. The loss and reexpression of RT6 may be related to activation and/or differentiation of T cells.