Substance P and neurokinin A both potentiated N-methyl-D-aspartate (NMDA)-induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca2+]i in these cells. Substance P, but not neurokinin A, increased [Ca2+]i in a subpopulation of neurons. The increase in [Ca2+]i was found to be due to Ca2+ influx through voltage-sensitive Ca2+ channels. Substance P and neurokinin A also potentiated the increase in [Ca2+]i produced by NMDA, but not by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, or 50 mM K+. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca2+]i can be dissociated from their effects on NMDA receptors.