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Bacteriophage integrase-directed insertion of transgenic constructs into specific genomic loci has been widely used byDrosophilacommunity. TheattP40landing site located on the second chromosome gained popularity because of its high inducible transgene expression levels. Here, unexpectedly, we found that homozygousattP40chromosome disrupts normal glomerular organization of Or47b olfactory receptor neuron (ORN) class inDrosophila. This effect is not likely to be caused by the loss of function ofMsp300, where theattP40docking site is inserted. Moreover, the attP40background seems to genetically interact with the second chromosomeOr47b-GAL4driver, which results in a similar glomerular defect. Whether the ORN phenotype is caused by the neighboring genes aroundMsp300locus in the presence ofattP40-based insertions or a second unknown mutation in theattP40background remains elusive. Our findings tell a cautionary tale about using this popular transgenic landing site, highlighting the importance of rigorous controls to rule out theattP40landing site-associated background effects.