Amino acid residues associated with functional specificity of cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK-like kinases (CLKs), which are collectively termed the CMGC group, were identified by categorizing and quantifying the selective constraints acting upon these proteins during evolution. Many constraints specific to CMGC kinases correspond to residues between the N-terminal end of the activation segment and a CMGC-conserved insert segment associated with coprotein binding. The strongest such constraint is imposed on a "CMGC-arginine" near the substrate phosphorylation site with a side chain that plays a role both in substrate recognition and in kinase activation. Two nearby buried waters, which are also present in non-CMGC kinases, typically position the main chain of this arginine relative to the catalytic loop. These and other CMGC-specific features suggest a structural linkage between coprotein binding, substrate recognition, and kinase activation. Constraints specific to individual subfamilies point to mechanisms for CMGC kinase specialization. Within casein kinase 2alpha (CK2alpha), for example, the binding of one of the buried waters appears prohibited by the side chain of a leucine that is highly conserved within CK2alpha and that, along with substitution of lysine for the CMGC-arginine, may contribute to the broad substrate specificity of CK2alpha by relaxing characteristically conserved, precise interactions near the active site. This leucine is replaced by a conserved isoleucine or valine in other CMGC kinases, thereby illustrating the potential functional significance of subtle amino acid substitutions. Analysis of other CMGC kinases similarly suggests candidate family-specific residues for experimental follow-up.