Several second-messenger-regulated protein kinases have been implicated in the regulation of N-methyl-D-aspartate (NMDA) channel function. Yet the role of calcium and cyclic-nucleotide-independent kinases, such as casein kinase II (CKII), has remained unexplored. Here we identify CKII as an endogenous Ser/Thr protein kinase that potently regulates NMDA channel function and mediates intracellular actions of spermine on the channel. The activity of NMDA channels in cell-attached and inside-out recordings was enhanced by CKII or spermine and was decreased by selective inhibition of CKII. In hippocampal slices, inhibitors of CKII reduced synaptic transmission mediated by NMDA but not AMPA receptors. The dependence of NMDA receptor channel activity on tonically active CKII thus permits changes in intracellular spermine levels or phosphatase activities to effectively control channel function.