Influx of Ca2+ through NMDA channels may initiate the stabilization of coactive synapses during development of the retinotectal projection in frogs. Ca2+ imaging techniques were applied to cultured tectal cells to investigate whether excitatory amino acids cause a rise in [Ca2+]i. High [K+], NMDA, and glutamate increase [Ca2+]i in about 75% of the cells. NMDA and glutamate responses were completely blocked in the absence of extracellular Ca2+ and by the NMDA receptor or channel blockers APV and MK-801. The NMDA response was also blocked by Mg2+. Quisqualate and kainate produced little or no rise in [Ca2+]i. These studies indicate that when tectal cells are exposed to the retinal ganglion cell transmitter glutamate, the predominant means of Ca2+ entry is through NMDA channels.