Extensive molecular, genetic, and anatomical analyses have suggested that olfactory memory is stored in the mushroom body (MB), a higher-order olfactory center in the insect brain. The MB comprises three subtypes of neurons with axons that extend into different lobes. A recent functional imaging study has revealed a long-term memory trace manifested as an increase in the Ca(2+) activity in an axonal branch of a subtype of MB neurons. However, early memory traces in the MB remain elusive. We report here learning-induced changes in Ca(2+) activities during early memory formation in a different subtype of MB neurons. We used three independent in vivo and in vitro preparations, and all of them showed that Ca(2+) activities in the axonal branches of alpha'/beta' neurons in response to a conditioned olfactory stimulus became larger compared with one that was not conditioned. The changes were dependent on proper G-protein signaling in the MB. The importance of these changes in the Ca(2+) activity of alpha'/beta' neurons during early memory formation was further tested behaviorally by disrupting G-protein signaling in these neurons or blocking their synaptic outputs during the learning and memory process. Our results suggest that increased Ca(2+) activity in response to a conditioned olfactory stimulus may be a neural correlate of early memory in the MB.