Timely deactivation of kinase cascades is crucial to the normal control of cell signaling and is partly accomplished by protein phosphatase 2A (PP2A). The catalytic (alpha) subunit of the serine-threonine kinase casein kinase 2 (CK2) bound to PP2A in vitro and in mitogen-starved cells; binding required the integrity of a sequence motif common to CK2 alpha and SV40 small t antigen. Overexpression of CK2 alpha resulted in deactivation of mitogen-activated protein kinase kinase (MEK) and suppression of cell growth. Moreover, CK2 alpha inhibited the transforming activity of oncogenic Ras, but not that of constitutively activated MEK. Thus, CK2 alpha may regulate the deactivation of the mitogen-activated protein kinase pathway.