A mouse monoclonal antibody (mAb) to the rat interleukin-2 receptor β (IL-2Rβ) chain was generated using IL-2Rβ cDNA-transfected mouse L929 cells for immunization and differential screening. This antibody, called L316, detects a cell surface protein with an apparent molecular mass of about 80 kDa. In peripheral lymphoid organs of young adult rats, IL-2Rβ expression is restricted to T and natural killer (NK) cells, and less than 10% of IL-2Rβ+ cells co-express the IL-2Rα chain. IL-2Rβ was detected on all NKRP-1hi (NK−) and NKRP-1lo cells (T-lineage cells of unknown function), most peripheral γδ T cells and on 30–40% of CD8 and 10% of CD4 αβ T cells. In the adult rat thymus, mAb L316 detects a small subset (about 1%) of predominantly IL-2Rα− cells which express cell surface markers characteristic of mature T lymphocytes and contain a high proportion of CD4−8− and CD4−8+ αβ T cell receptor (TCR)+ thymocytes. TCR-V usage suggests that major histocompatibility complex (MHC) class I plays a more important role than MHC class II in the selection of these cells. On immature CD4+8+ rat thymocytes, IL-2Rβ cell surface expression is readily induced by TCR stimulation in vitro, supporting the idea that in vivo, the IL-2Rβ+ phenotype is the result of TCR engagement during thymic selection.