Courtship conditioning assay can be used for measuring Drosophila memory. In this assay, male flies having courted unreceptive non-virgin females subsequently suppress their courtship behavior even toward receptive virgin females. This experience-dependent courtship suppression is based on memory formation. There are at least two phases of courtship memory as follows: (1) short-lasting memory (SLM), which lasts for several minutes or hours, and (2) long-term memory (LTM), which is maintained for several days. Since LTM formation depends on newly protein synthesis during and/or after conditioning, it is possible that transcriptional factors play a crucial role in LTM formation. The aterous (ap) gene encodes a LIM homeodomain transcriptional factor, which is well conserved from vertebrate to invertebrate. Previously, we demonstrated that ap mutants show defective courtship LTM. In this current study, we identified ap-expressing neurons in the brain which are involved in courtship LTM. First, we used flies with a GFP fusion knock-in allele of ap (ap::GFP) and observed their GFP-expressing neurons. GFP should be expressed in a pattern consistent with expression of the endogenous ap mRNA in this knock-in allele. GFP expression was detected in kenyon cells in the mushroom bodies (MBs) and several neuropeptide-expressing neurons (e.g., pigment-dispersing factor (PDF)-, neuropeptide F (NPF)-, and Leucokinin-producing neurons). Targeted expression of ap RNAi in the MB-, PDF-, or NPF-neurons induced defective courtship LTM. In addition, we confirmed that the LTM phenotype in ap mutant flies is rescued by GAL4-driven wild-type ap expression in the brain neurons containing MB-, PDF-, and NPF-neurons. Thus, our results suggest that ap expression in MB-, PDF-, and NPF-neurons is necessary and sufficient for courtship LTM.