The mushroom bodies, bilaterally symmetric regions in the insect brain, play a critical role in olfactory associative learning. Genetic studies in Drosophila suggest that plasticity underlying acquisition and storage of memory occurs at synapses on the dendrites of mushroom body Kenyon cells (Dubnau et al., 2001). Additional exploration of the mechanisms governing synaptic plasticity contributing to these aspects of olfactory associative learning requires identification of the receptors that mediate fast synaptic transmission in Kenyon cells. To this end, we developed a culture system that supports the formation of excitatory and inhibitory synaptic connections between neurons harvested from the central brain region of late-stage Drosophila pupae. Mushroom body Kenyon cells are identified as small-diameter, green fluorescent protein-positive (GFP+) neurons in cultures from OK107-GAL4;UAS-GFP pupae. In GFP+ Kenyon cells, fast EPSCs are mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors (nAChRs). The miniature EPSCs have rapid rise and decay kinetics and a broad, positively skewed amplitude distribution. Fast IPSCs are mediated by picrotoxin-sensitive chloride conducting GABA receptors. The miniature IPSCs also have a rapid rate of rise and decay and a broad amplitude distribution. The vast majority of spontaneous synaptic currents in the cultured Kenyon cells are mediated byalpha-bungarotoxin-sensitive nAChRs or picrotoxin-sensitive GABA receptors. Therefore, these receptors are also likely to mediate synaptic transmission in Kenyon cells in vivo and to contribute to plasticity during olfactory associative learning.