AbstractAbout 150 clock neurons are clustered in different groups in the brain of Drosophila. Among these clock neurons, some pigment‐dispersing factor (PDF)‐positive and PDF‐negative lateral neurons (LNs) are principal oscillators responsible for bouts of activity in the morning and evening, respectively. The full complement of neurotransmitters in these morning and evening oscillators is not known. By using a screen for candidate neuromediators in clock neurons, we discovered ion transport peptide (ITP) and short neuropeptide F (sNPF) as novel neuropeptides in subpopulations of dorsal (LNds) and ventral (s‐LNvs) LNs. Among the six LNds, ITP was found in one that coexpresses long neuropeptide F (NPF) and cryptochrome. We detected sNPF in two LNds that also express cryptochrome; these cells are distinct from three LNds expressing NPF. Thus, we have identified neuropeptides in five of the six LNds. The three LNds expressing cryptochrome, with either ITP or sNPF, are the only ones with additional projections to the accessory medulla. Among the five s‐LNvs in the adult brain, ITP was detected in the fifth neuron that is devoid of PDF and sNPF in the four neurons that also express PDF. By using a choline acetyltransferase (Cha) Gal4, we detected Cha expression in the two sNPF producing LNds and in the fifth s‐LNv. In the larval brain, two of the four PDF‐producing s‐LNvs coexpress sNPF. Our findings emphasize that the LNds are heterogeneous both anatomically and with respect to content of neuropeptides, cryptochrome, and other markers and suggest diverse functions of these neurons. J. Comp. Neurol. 516:59–73, 2009. © 2009 Wiley‐Liss, Inc.