Anti-idiotypic antibodies made against antigen-binding receptors on T lymphocytes with specificity for certain Ag-B locus antigens selectively react with T lymphocytes with potential immune reactivity against the very same Ag-B antigens. This was shown by affinity chromatography of normal Lewis T lymphocytes on anti-Ig columns after contact with the relevant anti-idiotypic antiserum. Here, it could be shown that incubation of the cells with an anti-(Lewis-anti-BN) antiserum caused subsequent selective retention of potential graft-vs.-host (GvH)-reactive cells against BN on the anti-Ig column, whereas Lewis T cells with reactivity against DA or August (Au) (carrying distinct Ag-B antigens in comparison to BN) passed through. The retained cells could be eluted and shown to display highly increased reactivity against BN with virtually no reactivity left against DA or Au antigens. Analogous results were obtained using an anti-(Lewis-anti-DA) antiserum. The anti-idiotypic antibodies can be used in fluorescent antibody tests to directly visualize the idiotype-positive cells. Using the separation design described above we analyzed selectively enriched or deleted T lymphocytes for presence of idiotypic cells as well as specific GvH reactivity. A highly significant positive correlation was found between percentage of a given idiotype in a population of T cells and the relevant GvH potential of the same T cells that can be visualized are indeed the very same T cells that express immune reactivity against the expected antigens. The present data would thus directly demonstrate the existence of a largely nonoverlapping population of immunocompetent T cells capable of reacting against the various Ag-B locus antigens in the rat. Highly purified, functionally intact immunocompetent T lymphocytes with restricted immune reactivity can thus be produced from normal lymphocyte populations for further analysis.