Living coronal brain slices were prepared from the rat prefrontal cortex (pFC) to determine the topographical distribution of the functional calcium-permeable N-methyl-D-aspartate (NMDA) receptor in vitro using calcium macroimage analysis with fura-2 AM. NMDA (0.25, 0.5, 1 or 2 x 10(-4) M for 30 s) bath application showed a dose-dependent [Ca2+]i increase from the steady-state level in several portions selected from the whole hemisphere slice. This regional response was suppressed by D-2-amino-5-phosphonopentanoic acid (10(-4)M) and reinforced by glycine coperfusion (5 x 10(-6) M). Image analysis showed that the cluster responsive to NMDA lay locally on the medial or lateral pFC even in the presence of tetrodotoxin.