Fluorine closely mimics the steric requirement of hydrogen at enzyme receptor sites, but its strong electronegativity significantly alters the reactivity of neighboring centers. Therefore, point-fluorination of biologically active molecules is an important technology with which to investigate the relationship between a biologically active compound with a receptor protein. We synthesized point-fluorinated pheromone analogues of eldanolide and measured their biological activity by electroantennography (EAG) to understand the importance of conformation in the specificity of ligand recognition by the olfactory receptor. By comparing EAG activities and conformational analysis of these molecules using density functional theory calculations, significant differences were found in the population of preferable conformers between EAG-active compounds and EAG-inactive compounds. Based on these results, we propose a working hypothesis for the possible origin of the diversity of relationship between enantiomer and activity in pheromone perception response. These results obtained in the investigation of the mechanism of chemical communication through pheromone molecules among insects should be useful to expand the horizons of medicinal chemists.