To determine whether presynaptic input is necessary for postsynaptic differentiation, we isolated hippocampal neurons in microisland culture and thus deprived pyramidal cells of GABA input and GABAergic neurons of glutamate input. We find that glutamate input is necessary for clustering the AMPA-type glutamate receptor but not for clustering the NMDA receptor or the associated PSD-95 family scaffold in GABAergic cells; GABA input is not necessary for clustering the GABA A receptor or gephyrin in pyramidal cells. Isolated neurons showed a surprising mismatch of presynaptic and postsynaptic components. For example, in isolated pyramidal neurons, although GABA A receptor clusters covered Ͻ4% of the dendritic surface and presynaptic boutons covered Ͻ12%, a full two-thirds of the GABA A receptor clusters were localized inappropriately opposite the non-GABAergic, presumed glutamatergic, terminals. Furthermore, inhibitory and excitatory postsynaptic components were segregated into separate clusters in isolated cells and apposed to separate boutons of a single axon. Thus, GABA A receptors were clustered opposite some terminals, whereas NMDA receptors were clustered opposite other terminals of a single axon. These results suggest the involvement of a synaptogenic signal common to glutamate and GABA synapses that permits experimentally induced mismatching of presynaptic and postsynaptic components in isolated neurons, as well as a second specificityconferring signal that mediates appropriate matching in mixed cultures.Key words: synaptogenesis; hippocampus; neuron culture; glutamate receptor; GABA receptor; PSD-95; gephyrin; autapse Synapse formation requires the aggregation of receptors and associated signaling molecules in dendrites in precise apposition to aggregates of synaptic vesicles and release machinery in axons. The clustering of neurotransmitter receptors in the postsynaptic membrane has been well studied at the neuromuscular junction, where acetylcholine receptors cluster at the muscle end plate (for review, see Sanes and Lichtman, 1999). Although acetylcholine receptors can cluster spontaneously on the muscle surface, nerve input induces a redistribution of receptors to generate local clustering and stabilization beneath the nerve terminal. It has been known for some time that similar postsynaptic receptor aggregates are present at CNS synapses (Triller et al., 1985;Somogyi et al., 1989;Craig et al., 1993), but the process by which these aggregates form and the role of presynaptic input are not well understood. In addition, the multiplicity of transmitter systems and the problem of segregating receptors to appropriate postsynaptic sites further complicate receptor clustering in neurons.Previous studies have shown that presynaptic and postsynaptic specializations for synapses using the neurotransmitters GABA and glutamate are appropriately matched in hippocampal neurons. Thus, AMPA-and NMDA-type glutamate receptors along with the excitatory synapse-associated molecules of the PSD-95 family, GKAP/SAPAP, ␣-actin...