The fruitfly, Drosophila, is dependent on its olfactory sense in food search and reproduction. Processing of odorant information takes place in the antennal lobes, the primary olfactory center in the insect brain. Besides classical neurotransmitters, earlier studies have indicated the presence of a few neuropeptides in the olfactory system. In the present study we made an extensive analysis of the expression of neuropeptides in the Drosophila antennal lobes by direct profiling using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry and immunocytochemistry. Neuropeptides from seven different precursor genes were unambiguously identified and their localization in neurons was subsequently revealed by immunocytochemistry. These were short neuropeptide F, tachykinin related peptide, allatostatin A, myoinhibitory peptide, SIFamide, IPNamide, and myosuppressin. The neuropeptides were expressed in subsets of olfactory sensory cells and different populations of local interneurons and extrinsic (centrifugal) neurons. In some neuron types neuropeptides were colocalized with classical neurotransmitters. Our findings suggest a huge complexity in peptidergic signaling in different circuits of the antennal lobe.