A dissociated cell culture from the postnatal rat retina was established to characterize the synapses formed by retinal ganglion neurons (RGNs) in vitro. An antibody against Thy-1.1 was used to preselect putative RGNs for pair patch-clamp recording with the principal aim of identifying the released transmitter(s) and estimating the role of different types of voltage-activated Ca2+ channels in evoked transmitter release. The population of Thy-1+ neurons was heterogeneous. Staining patterns, soma-dendritic geometries and axon length displayed variations that could be related to basic electrophysiological properties, such as amplitudes of voltage-activated Na+ currents (INa(V)), action potential size and capacity for repetitive discharge. Out of 73 coupled connections, 33 pairs were glutamatergic. With no exception, these connections were formed by the axons of strongly labeled Thy-1+ neurons with large INa(V) (typically > 2 nA) and repetitive firing over a broad current range. Such neurons were classified as RGNs. Forty out of 73 coupled pairs were GABAergic. These connections were always formed by weakly stained Thy-1+ neurons with small INa(V) (typically < 2 nA) and very limited capacity for repetitive discharge. Such neurons were tentatively classified as displaced amacrine cells. Evoked EPSCs in response to RGN activation were completely blocked by low concentrations of Cd2+ or Gd3+. omega-CgTx-GVIA (5 microM) reduced EPSCs to 67 +/- 29%, omega-AgaTx-IVA (200 nM) had no effect, and nifedipine (15 microM) enhanced the evoked EPSCs. Our experiments indicate that (1) the transmitter released by RGNs is glutamate and (2) the major part of synaptic glutamate release is governed by a novel toxin-resistant Ca2+ channel. The results further suggest that the characteristic phenotype of RGNs is well maintained in dissociated cell culture. In conjunction with electrophysiological tests Thy-1+ labeling can be used for RGN identification.