Multiple-site optical recording of transmembrane voltage activity, using a voltage-sensitive dye (NK2761) and a 12 x 12 element photodiode array, was employed to monitor neural responses in the slice preparations that were dissected from 6–9-d-old embryonic brainstems. Transmembrane voltage-related optical responses evoked by a brief square current pulse applied with a microsuction electrode to the vagus nerve were recorded simultaneously from many sites in the preparation. In preparations from 6-d-old embryos, only action potential-related spike-like optical signals were often detected in the normal bathing solution containing 0.5 mM Mg2+. However, in several loci of some preparations, excitatory postsynaptic potential-related slow signals were elicited in a Mg(2+)-free bathing medium, and these elicited slow signals were blocked by DL-2-amino-5-phosphonovaleric acid (2-APV). These results show a possible embryonic expression of NMDA receptor- mediated excitatory postsynaptic potentials. The slow signals were usually detected from tested preparations from 7–9-d-old embryos in normal bathing solution. The later phase of the slow signals was enhanced in a Mg(2+)-free bathing medium, and the enhanced component was also sensitive to 2-APV and insensitive to 6-cyano-7- nitroquinoxaline-2,3-dione (CNQX). There were regional differences in the degree of the enhancement of the slow signals in the Mg(2+)-free bathing medium, and of the 2-APV-induced reduction of the slow signals. We have constructed early developmental maps of the spatial patterns of the slow signals related to Mg(2+)- and 2-APV-sensitive postsynaptic potentials. These maps reflect the spatial distribution of functional expression of the NMDA receptors during early development of the chick brainstem. The present findings are new data that have not been reported previously, and they have been obtained for the first time using the multiple-site optical recording technique with a voltage- sensitive dye.