Although both the CD4 and CD8 molecules enhance antigen responsiveness mediated by the T cell receptor (TCR), it is not known whether CD4 and CD8 initiate similar or different intraceUular signals when they act as coreceptors. To characterize the early signals transmitted by CD4 and CDS, both CD4 and CDSo- were expressed in the same murine T cell hybridoma. In the double positive transfectants, CD4 and CD8 associated with equal amounts of p56 Ick (Lck), and both molecules enhanced interlenkin 2 (IL-2) production equivalently when crosslinked with suboptimal levels of anti-TCR antibody. However, in an in vitro kinase assay, crosslinking CD4 initiated fourfold greater kinase activity compared with CD8 eross-linking. In the same assay, when CD4 or CD8 was cross-linked to the TCR, novel phosphorylated proteins were found associated with the TCR/CD4 complex but not with the TCR/CD8 complex. Consistent with this data, antiphosphotyrosine immunoblotting revealed greater tyrosine phosphorylation ofintracellular substrates after TCIL/CD4 cross-linking compared with TCR/CD8 cross-linking. Additionally, a specific protein kinase C inhibitor (RO318220) inhibited CDSmediated enhancement of IL-2 production far more effectively than CD4-mediated enhancement. Thus, it appears that CD8α may depend more on a protein kinase C-mediated signaling pathway, whereas CD4 may rely on greater tyrosine kinase activation. Such differential signaling via CD4 and CD8 has implications for thymic ontogeny and T cell activation.