Biogenic amines, such as serotonin and dopamine, can be important in reinforcing associative learning. This function is evident as changes in memory performance with manipulation of either of these signals. In the insects, evidence begins to argue for a common role of dopamine in negatively reinforced memory. In contrast, the role of the serotonergic system in reinforcing insect associative learning is either unclear or controversial. We investigated the role of both of these signals in operant place learning in Drosophila. By genetically altering serotonin and dopamine levels, manipulating the neurons that make serotonin and dopamine, and pharmacological treatments we provide clear evidence that serotonin, but not dopamine, is necessary for place memory. Thus, serotonin can be critical for memory formation in an insect, and dopamine is not a universal negatively reinforcing signal.