We sampled atmospheric barn air using a volumetric air sampler in ten barns near Cooperstown, NY and six barns near Rochester, MN, and, with radioimmunoassays, measured allergens of Aspergillus fumigatus, Thermoactinomyces vulgaris, Micropolyspori faeni, short ragweed, rye grass group I pollen, Alternaria (Alt-1), Dermatophagoides sp. Lepidoglyphus destructor, common insect allergen, mouse urine, rat urine, and cattle epithelium. The most abundant allergen present was A. fumigatus followed by L. destructor. This study provides initial data on barn aerobiology and demonstrates for the first time the abundance of L. destructor allergens in North American dairy barns. More comprehensive study of barns, poultry houses, confinement houses for swine, and other agricultural environments from various geographic locations is needed to define the allergen levels to which millions of farm workers are exposed each day. While most of the allergens were expected, the presence of airborne allergens reactive with antisera to Dermatophagoides suggests indirectly that substantial amounts of pyroglyphid mites are present in some barns.