An enzyme-linked immunosorbent assay (ELISA) for the olive fruit fly pheromone, Bactrocera oleae Gmelin, was developed. The assay uses polyclonal antibodies, raised in rabbits, against (+/-)-beta-[3-(1,7-dioxaspiro[5.5]undecane)]propionic acid, 2 (hapten I), conjugated to the KLH (keyhole limpet hemocyanin) by the carbodiimide method. A second hapten, (+/-)-delta-[3-(1,7-dioxaspiro[5.5]undecane)]butylamine, 3 (hapten II), after conjugation to a biotin moiety, was used for indirect immobilization onto ELISA microwells precoated with the glycoprotein avidin. The developed ELISA method measures the synthetic olive fruit fly pheromone in concentrations ranging between 0.08 and 10 microg/mL and shows great promise for practical applications for pheromone detection in environmental and biological samples. The results obtained strongly indicate that this technique, to our knowledge the first insect pheromone enzyme-linked immunosorbent assay so far reported, is a fast, sensitive, inexpensive, and highly convenient method for the analysis of a volatile and low molecular weight compound such as 1,7-dioxaspiro[5.5]undecane, 1.