Monospecific rabbit antibodies were used to develop a sensitive two-site enzyme immunoassay to measure a major dog hair and dander allergen, Can f I. This Can f I assay demonstrated no reaction with 17 heterologous allergen sources, including dog albumin, cat, guinea pig, and horse. Analysis of serial dilutions of purified Can f I and the international standard for dog was parallel. The assay was considered specific for Can f I with a lower limit of detection at 0.03 micrograms/ml. Total imprecision was from 2% to 6%. Commercial dog extracts for specific immunotherapy contained from 0.7 to 290 micrograms of Can f I per milliliter. The assay was used to measure Can f I in 136 house dust samples collected from 103 homes across the United States. Concentration of the dog allergen was expressed as micrograms of Can f I per gram of dust. Prevalence of Can f I in the dust samples ranged from less than 0.3 to 10,000 micrograms/gm. Serial dilutions of samples containing Can f I were parallel to the standard. The median Can f I value for homes with a dog in residence was 120 micrograms/gm, and for homes with no dog, 3 micrograms/gm. With few exceptions, homes with no dog in residence had less than 10 micrograms/gm. This Can f I assay will provide useful information for assessing commercial extracts as well as monitoring dog-allergen exposure and allergen-control methods.