BACKGROUND: Few studies have addressed exposure and sensitization to mite allergens in Andean countries. OBJECTIVES: To identify the main mite species in 3 locations at different altitudes in Ecuador and to verify skin test reactivity to various mite species in allergic individuals in Quito, Ecuador. METHODS: Mattress dust samples were collected in Quito (2,800 m above sea level), Cuenca (2,500 m above sea level), and Guayaquil (sea level). Mite species present in the samples were isolated, identified, and counted. Der p 1 and Der f 1 levels were measured using monoclonal antibody-based enzyme immunoassays. Four hundred thirty-five patients in Quito diagnosed as having allergic rhinitis or asthma underwent skin testing with commercial extracts of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Tyrophagus putrescentiae, and Lepidoglyphus destructor. In addition, Glycyphagus domesticus, Acarus siro, and Aleuroglyphus ovatus were tested in 362, 262, and 279 patients, respectively. RESULTS: Twenty-one mite species were identified. Large populations of mites were detected above 2,500 m of altitude. All the dust samples contained detectable levels of Der p 1 or Der f 1. Positive skin prick test reactions to D. pteronyssinus, D. farinae, B. tropicalis, L. destructor, T. putrescentiae, A. ovatus, A. siro, and G. domesticus were obtained in 60.9%, 56.8%, 17.0%, 19.3%, 10.6%, 15.8%, 8.8%, and 11.0% of the patients, respectively. CONCLUSIONS: Most analyzed mattresses contained several species of mites. Mite allergen levels were high. This study confirms the importance of house dust and storage mite allergens in Ecuador in areas above 2,500 m of altitude, where humidity remains high year round.