The American house dust mite, Dermatophagoides farinae Hughes, and European house dust mite, Dermatophagoides pteronyssinus Trouessart, are major pests of medical importance throughout the developed world, causing atopic diseases such as asthma, rhinitis, and atopic dermatitis. Previous studies in our laboratory have shown that the behavioral responses of house dust mites toward volatiles from food sources could be assessed using a Y-tube olfactometer assay. The current study used this Y-tube assay to investigate house dust mite pheromones. A hexane extract of D. farinae, along with fractions of the extract prepared by microscale liquid chromatography over Florisil, were tested for behavioral activity. One of the chromatographic fractions was shown to be significantly attractive (P < 0.05) for D. farinae, compared with a solvent control. Coupled gas chromatography-mass spectrometry analysis of this behaviorally active fraction indicated that neryl or geranyl formate was the major component. Peak enhancement by gas chromatography, using authentic samples of the neryl and geranyl isomers prepared in high purity by chemical synthesis, confirmed the identity of the major peak as neryl formate. In Y-tube assays, male and female D. farinae and D. pteronyssinus both were significantly attracted to synthetic neryl formate at doses of 100 and 10 ng, respectively (P < 0.05). No significant differences were found for D. farinae and D. pteronyssinus when synthetic neryl formate and house dust mite extracts containing natural neryl formate were tested at the same level. Dynamic headspace collection of D. farinae and D. pteronyssinus colonies showed that neryl formate was released as a volatile organic compound by both species. Our study shows that neryl formate is an aggregation pheromone for D. farinae and D. pteronyssinus, and has the potential to be used as part of a novel lure-and-kill system for house dust mite control.