Intracellular regulation of NMDA-receptor-mediated currents in cultured rat spinal dorsal horn neurons was investigated by means of simultaneously recording whole-cell currents and intracellular Ca2+ concentration ([Ca2+]i). During recordings in which EGTA (11 mM) was used to buffer intracellular Ca2+, NMDA currents showed 'rundown'; the amplitude of the currents gradually declined to a stable level approximately 50% of the initial level within 15 min of the beginning of recording. In these experiments, the level of [Ca2+]i decreased rapidly once whole-cell recording was attained and baseline [Ca2+]i remained below 100 nM. Each NMDA current was associated with a transient increase in [Ca2+]i which was prevented when BAPTA (30 mM) was substituted for EGTA. However, inclusion of BAPTA in the intracellular solution failed to affect the rundown of the currents. In contrast, including Mg-ATP (4 mM) prevented the rundown of NMDA currents and resulted in an increase in the current amplitude. Thus, our results indicate that rundown of the NMDA currents is not due to raised [Ca2+]i and are consistent with regulation of NMDA currents by phosphorylation.