The leaf surface of most terrestrial plants is covered with plant hairs called trichomes. These epidermal appendages are thought to contribute to many aspects of plant defense against biotic and abiotic stresses in a variety of species. Trichome development has been intensively studied in Arabidopsis, and the phytochemical composition of trichomes was analyzed in a number of plant species. However, comparatively little is known of the proteins expressed. We therefore initiated a proteome approach to better define the cellular mechanisms operating in plant trichomes using two-dimensional gel electrophoresis to separate proteins of whole leaves and isolated trichomes. Tobacco was chosen due to the presence of glandular trichomes involved in the secretion of defense compounds. Comparative image analysis of the protein patterns indicated a number of spots, which were highly enriched in trichomes relative to leaves. These spots were excised for identification by mass spectrometry. The results showed that among the proteins specifically enriched in trichomes, the components of stress defense responses were strongly represented. The high expression of stress-related proteins was verified by Western blotting. Superoxide dismutase isoforms were additionally analyzed by activity staining. Our results demonstrate feasibility of the proteome approach to elucidate the cell biology of plant trichomes.