Negatively reinforced olfactory conditioning has been widely employed to identify learning and memory genes, signal transduction pathways and neural circuitry in Drosophila. To delineate the molecular and cellular processes underlying reward-mediated learning and memory, we developed a novel assay system for positively reinforced olfactory conditioning. In this assay, flies were involuntarily exposed to the appetitive unconditioned stimulus sucrose along with a conditioned stimulus odour during training and their preference for the odour previously associated with sucrose was measured to assess learning and memory capacities. After one training session, wild-type Canton S flies displayed reliable performance, which was enhanced after two training cycles with 1-min or 15-min inter-training intervals. Higher performance scores were also obtained with increasing sucrose concentration. Memory in Canton S flies decayed slowly when measured at 30 min, 1 h and 3 h after training; whereas, it had declined significantly at 6 h and 12 h post-training. When learning mutant t beta h flies, which are deficient in octopamine, were challenged, they exhibited poor performance, validating the utility of this assay. As the Drosophila model offers vast genetic and transgenic resources, the new appetitive conditioning described here provides a useful tool with which to elucidate the molecular and cellular underpinnings of reward learning and memory. Similar to negatively reinforced conditioning, this reward conditioning represents classical olfactory conditioning. Thus, comparative analyses of learning and memory mutants in two assays may help identify the molecular and cellular components that are specific to the unconditioned stimulus information used in conditioning.