Protein kinase C (PKC) potentiates NMDA receptors in hippocampal, trigeminal, and spinal neurons. Although PKC phosphorylates the NMDA receptor subunit NR1 at four residues within the C terminal splice cassette C1, the molecular mechanisms underlying PKC potentiation of NMDA responses are not yet known. The present study examined the role of Ca2+ in PKC potentiation of recombinant NMDA receptors expressed in Xenopus oocytes. We found that Ca2+ influx through PKC-potentiated NMDA receptors can further increase the NMDA response ("Ca2+ amplification"). Ca2+ amplification required a rise in intracellular Ca2+ concentration at or near the intracellular end of the channel and was independent of Ca2+-activated Cl- current. Ca2+ amplification depended on extracellular Ca2+ concentration during NMDA application and not during PKC activation. Ca2+ amplification was reduced by the membrane-permeant Ca2+-chelating agent BAPTA-AM. Mutant receptors with greatly reduced Ca2+ permeability did not exhibit Ca2+ amplification. Receptors containing the NR1 N-terminal splice cassette showed more Ca2+ amplification, possibly because of their larger basal current and therefore greater Ca2+ influx. Contrary to expectation, splicing out the two C-terminal splice cassettes of NR1 enhanced PKC potentiation in a manner independent of extracellular Ca2+. This observation indicates that PKC potentiation does not require phosphorylation of the C1 cassette of the NR1 subunit. PKC potentiation of NMDA receptors in vivo is likely to be affected by Ca2+ amplification of the potentiated signal; the degree of amplification will depend in part on alternative splicing of the NR1 subunit, which is regulated developmentally and in a cell-specific manner.