The discovery that neuroligin is a key protein involved in synapse formation offers the unprecedented opportunity to induce functional synapses between neurons and heterologous cells. We took this opportunity recording for the first-time synaptic currents in human embryonic kidney 293 (HEK293) cells transfected with neuroligin and the N-methyl-d-aspartate or AMPA receptor subunits in a co-culture with rat cerebellar granule cells. These currents were similar to synaptic currents recorded in neurons, and their decay kinetics was determined by the postsynaptic subunit combination. Although neuroligin expression was sufficient to detect functional synapses, cotransfection of HEK293 cells with Postsynaptic density-95/synapse-associated protein-90 (PSD-95) significantly increased current frequency. Our results support the central role of neuroligin in the formation of CNS synapses, validate the proposal that PSD-95 allows synaptic maturation, and provide a unique experimental model to study how molecular components determine functional properties of excitatory synapses.