AbstractDrosophila melanogaster produces fatty acid amides, and thus, provides a model to unravel the pathways for their biosynthesis. We previously demonstrated that arylalkylamine N‐acetyltransferase‐like 2 (AANATL2) from D. melanogaster will catalyze the formation of long‐chain N‐acylserotonins and N‐acyldopamines in vitro. Generating silencing RNA via the UAS/GAL4 bipartite approach for targeted gene expression effectively decreased the endogenous levels of the AANATL2 transcripts in D. melanogaster, as shown by reverse transcription quantitative polymerase chain reaction. Consistent with these data, western blot analysis of the offspring of the AANATL2 knockdown flies using an anti‐AANATL2 antibody revealed a significant reduction in the expression of the AANATL2 protein. Reduced expression of AANATL2 decreased the cellular levels of N‐palmitoyldopamine (PALDA), providing strong evidence that AANATL2 is responsible for the biosynthesis of PALDA in vivo. This is the first time that the expression of an AANAT has been reduced in D. melanogaster to link one of these enzymes to the in vivo production of an N‐acylarylalkylamide.