The two-spotted mite (TSM) is commonly found on fruit trees, herbaceous plants, and greenhouse flowers. However, a recent investigation indicated that the sensitization rate to TSM was as high as that of house-dust mites (HDMs) in nonfarmers as well as in farmers working in orchards in this country. The aim of this study was to identify immunoglobulin E (IgE)-binding components within TSM and to evaluate the allergenic relationship with the citrus red mite (CRM) and HDM. Sera were collected from eight patients who were not farmers and who had asthma with high serum-specific IgE to the TSM and from unexposed controls showing negative responses to the TSM on skin-prick test. Twelve percent sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis were applied. To evaluate allergenic relationships with HDMs and CRMs, two kinds of sera pools were used: one (A) showing positive responses to both TSMs and HDMs and the other (B) showing an isolated positive response to TSMs. ELISA inhibition tests using A and B pooled sera were conducted. The TSM-ELISA inhibition test using sera A showed significant inhibition with addition of TSMs and CRMs, partial inhibition with HDMs, and minimal inhibition with other inhalant allergens. The ELISA inhibition test using sera B showed significant inhibition with TSMs and CRMs and minimal inhibition was noted with HDMs as well as other inhalant allergens. Immunoblot analysis using individual sera showed seven IgE-binding components (75, 56, 47, 41, 37, 28, and 14 kDa) and two (75 and 14 kDa) of them were bound to IgE in > 50% of the sera tested. Seven IgE-binding components were identified within the TSM extract and two (75 and 14 kDa) could be considered major allergens. It is suggested that the TSM contains species-specific allergen in addition to shared allergens with CRMs and HDMs.