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 intracellular signals when they act as coreceptors. To characterize the early signals transmitted by CD4 and CD8, both CD4 and CD8 alpha were expressed in the same murine T cell hybridoma. In the double positive transfectants, CD4 and CD8 associated with equal amounts of p56lck (Lck), and both molecules enhanced interleukin 2 (IL-2) production equivalently when cross-linked with suboptimal levels of anti-TCR antibody. However, in an in vitro kinase assay, cross-linking CD4 initiated fourfold greater kinase activity compared with CD8 cross-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 of intracellular substrates after TCR/CD4 cross-linking compared with TCR/CD8 cross-linking. Additionally, a specific protein kinase C inhibitor (RO318220) inhibited CD8-mediated enhancement of IL-2 production far more effectively than CD4-mediated enhancement. Thus, it appears that CD8 alpha 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.