The CD8 molecule plays an important role in the differentiation of CD8+ T cells in the thymus and in their normal function in the periphery. CD8 exists on the cell surface in two forms, the αα homodimer and the αβ heterodimer. Recent studies indicate an important role for the CD8β chain in thymic development of CD8+ T cells and suggest that signaling via CD8αβ may be distinct from CD8αα. To better understand these differences, we introduced the CD8β gene into a T cell hybridoma which only expressed the CD8αα homodimer. In the parent hybridoma, cross-linking of the CD8α chain led to minimal enhancement of CD8-associated Lck tyrosine kinase activity. In the CD8β+ transfectants, several observations suggested that CD8β modifies CD8α-associated Ink tyrosine kinase activity: (a) in in vitro kinase assays, antibodymediated crosslinking of CD8 alone, or CD8 cross-linking with the TCR, resulted in 10-fold greater activation of Lck kinase activity, compared to cells expressing CD8αα alone; (b) in vivo, markedly enhanced tyrosine phosphorylation of several intracellular proteins was observed upon CD8 cross-linking with the TCR in CD8αβ-expressing cells, compared to cells expressing CD8αα alone; and (c) Ink association with CD8β was stabilized by the coexpression of CD813. Thus, the differential Lck kinase activation and tyrosine phosphorylation seen with CD8αα vs. CD8αβ may reflect the unique signaling capabilities of the CD8β molecule. These differences in signaling may, in part, account for the diminished ability to generate CD8 single positive thymocytes in mice bearing a homozygous disruption of the CD8β gene.