CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.