Study of lymphocyte subsets has been facilitated by the detection of various surface markers that are related to distinct functions. We have explored the use of affinity methods for separation of large quantities (10(8)) of rat cells and used this technique to identify surface markers of cytotoxic T lymphocytes. Splenocytes from animals bearing skin allografts for 7 days were depleted of B cells and were separated into T cell subsets by using W3/25 and OX8 monoclonal antibodies, which mark two nonoverlapping populations of peripheral T cells. Our results demonstrate that the cytotoxic T cells are W3/25- and OX8+. They also bear minimal or no Ag-F alloantigen, a peripheral T cell marker, and they express Ia-like products analogous to those of the mouse I-A subregion but not of the I-E/C subregion.