The NMDA receptor is modulated by changes in the intracellular calcium concentration, through activation of various intracellular calcium-dependent proteins. We have investigated regulation of single NMDA receptor channel activity by the calcium-sensing proteins alpha-actinin and calmodulin. Both of these proteins bind to the NMDA receptor NR1 subunit C-terminus at the C0 region where they compete for occupation of the C0 site and contribute to calcium-dependent inactivation of NMDA receptor-mediated whole-cell currents. Calmodulin has also been shown to bind to the neighbouring C1 region where it has been shown to reduce single channel open time. To investigate regulation of single NMDA channel activity by alpha-actinin and calmodulin, we selected concentrations of these two proteins that would result in maximal binding to the C0 region and/or the C1 region in the case of calmodulin. Alpha-actinin binding was found to predominantly decrease single channel shut time, resulting in an increased open probability (Popen), whereas calmodulin binding reduced single channel mean open time, resulting in an overall reduction in Popen. The physiological implications of these findings are discussed.