CD5 attenuates TCR-induced signals in immature thymocytes but functions as a costimulatory molecule potentiating TCR/CD3-mediated activation in mature, peripheral T cells. We have recently shown that the serine/threonine kinase, casein kinase 2 (CK2), a major regulator of cell growth and signaling, associates with and is activated by CD5. Therefore, we tested the possibility that differential regulation of CK2 activity by CD5 may be associated with these differences in CD5 modulation of TCR signaling. Consistent with our hypothesis, CD5-specific cross-linking activated associated CK2 in thymocytes but not active in mature splenocytes. Differential regulation of CD5-associated CK2 provides, for the first time, a potential mechanism for the differential effects of CD5 signaling in immature and mature cells. We propose that CD5 modulates Ag receptor activity through developmentally regulated activation of CD5-associated CK2.