Background: Pollen is an important trigger of seasonal rhinitis, conjunctivitis, and/or allergic asthma, and an exacerbating factor in atopic dermatitis. Pollen grains contain allergen proteins, enzymes, and bioactive lipid mediators, the latter two possibly involved in the pathogenesis of allergic diseases through IgE-independent mechanisms. Methods: We analyzed the patterns of release of endopeptidases from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and birch, ragweed, and two grasses, Kentucky blue and cultivated rye, using synthetic substrates, class-specific inhibitors, and zymography. The proteins released were analyzed by gel electrophoresis. Eicosanoid-like substances were measured by enzyme-linked immunosorbent assays for prostaglandin E<sub>2</sub> and leukotriene B<sub>4</sub>. Results: Major fractions of proteins, eicosanoid-like substances, and at least one molecular species of serine endopeptidase were released into phosphate-buffered saline from the pollen grains at 37°C within 25 min or 60 min without sonication. In the Cupressaceae/Taxodiaceae family, sonication was necessary for the release of other proteins and another serine endopeptidase. In birch, ragweed, and the grasses, most of the serine and cysteine endopeptidases were released without sonication. Proteases released within 25 min digested gelatin and/or casein differently among plant species. Conclusions: Grains of allergenic pollen release proteases, which can digest not only short synthetic substrates but also protein substrates, along with eicosanoid-like substances and proteins. The release of these components could contribute to the formation of a microenvironment optimum for initiation of the sensitization or the exacerbation of pollen allergy in tissues exposed to pollen grains.