Mammalian central synapses commonly specialize in one fast neurotransmitter, matching the content of their presynaptic vesicles with the appropriate receptors in their postsynaptic membrane. Here, I show that hippocampal cultures contain autaptic glutamatergic synapses that contravene this rule: in addition to postsynaptic glutamate receptors, they also express clusters of functional postsynaptic GABA(A) receptors yet lack presynaptic GABA. Hence, these synapses are presynaptically silent with respect to GABA. They can be unsilenced by loading GABA into presynaptic vesicles by endocytosis, after which a postload IPSC appears. This IPSC is similar to native IPSCs recorded from GABAergic interneurons in the same cultures. Thus, these "mistargeted" GABA(A) receptors, which apparently lack a signal that confers synaptic specificity, function almost normally. After GABA loading, glutamatergic miniature postsynaptic currents acquire a slow tail that is mediated by GABA(A) receptors, showing that synaptic vesicles can accommodate both the usual concentration of native glutamate and a saturating concentration of loaded GABA. After brief Ca(2+)-dependent exocytosis, endocytosis of GABA can proceed in low-Ca(2+) external solution. The amplitude of the postload IPSC declines exponentially with repetitive stimulation as the endocytosed GABA passes through the presynaptic vesicle cycle and is depleted. Hence, by using GABA as an exogenous but physiological tracer, the properties of these presynaptically silent synapses can provide novel insights into the content and cycling of vesicles in presynaptic terminals.