Unselected CD4+8+ rat thymocytes, generated in vitro from their direct precursors, are readily converted to functional TCRhigh T cells by stimulation with immobilized TCR-specific mAb plus IL-2. Lineage decision invariably occurs toward CD4-8+, regardless of the timing of TCR stimulation after entry into the CD4+8+ compartment or the concentration of TCR-specific mAb used for stimulation. CD4-specific mAb synergizes with suboptimal TCR-specific mAb in inducing T cell maturation, but lineage decision remains exclusively CD4-8+. These results contrast with those obtained in mice, in which Abs to the TCR complex were shown to promote CD4+8- T cell maturation from CD4+8+ thymocytes. Surprisingly, when rat and mouse CD4+8+ thymocytes were stimulated with PMA/ionomycin under identical conditions, the opposite lineage commitment was observed, i.e., mouse thymocytes responded with the generation of CD4+8- and rat thymocytes with the generation of CD4-8+ cells. It thus seems that CD4+8+ thymocytes of the two species respond with opposite lineage decisions to strong activating signals such as given by TCR-specific mAb or PMA/ionomycin. A possible key to this difference lies in the availability of p56lck for coreceptor. supported signaling. We show that in contrast to mouse CD4+8+ thymocytes, which express both a complete and a truncated CD8 alpha-chain (CD8 alpha') unable to bind p56lck, rat thymocytes only express full-length CD8 alpha molecules. Mice, but not rats, therefore may use CD8 alpha' as a "dominant negative" coreceptor chain to attenuate the CD8 signal, thereby facilitating MHC class II recognition through the higher amount of p56lck delivered, and rats may use a different mechanism for MHC class distinction during positive selection.