Monoclonal antibodies (mAbs) that recognize particular structures have been widely used as molecular markers in various aspects of C. elegans studies. To obtain a new series of mAbs useful to characterize embryogenesis, we have developed a new method to efficiently generate hybridoma lines that produce organelle-, cell type-, and tissue-specific mAbs, in which <
sym08>subtracted<
sym09> embryonic extracts are used as antigens. This <
sym08>antigen subtraction<
sym09> method requires two rounds of hybridoma production. In the first round of hybridoma production, sonicated C. elegans embryos were used as antigens. Many hybridomas were shown to produce mAbs (526 immunofluorescence-positive wells and 184 Western blotting positive wells among 874 wells with hybridoma colonies). Immunofluorescence with most of them stained whole cytoplasm of all cells in the worm embryo. These antibodies appeared to recognize abundant proteins in every cell. In the second round, abundant antigens were removed from the embryonic extracts by immunoprecipitation with pools of hybridoma culture-supernatants obtained in the first round, which should contain mAbs against abundant, cell-type-non-specific antigens. The remaining fraction (=<
sym08>subtracted antigens<
sym09>) was expected to be enriched with less abuntant, organelle/cell/tissue-specific proteins, which was then used for immunization. In the second hybridoma production, 165 immunofluorescence-positive wells were obtained among 512 wells with hybridoma colonies. As expected, organelle/cell/tissue-specific mAbs were obtained with a much higher probability compared to the first round, and ~40 hybridoma lines (<
sym08>KT<
sym09> series) were established. The recognized organelles/cells/tissues include: P-granules, body wall muscles, a subset of epidermal cells, seam cells, pharynx, centrosomes, nuclear envelope and other undefined structures/positions. Thus, the <
sym08>antigen subtraction<
sym09> method is useful to obtain mAbs against less abundant components in biological specimen. Detailed spatio-temporal staining patterns of these mAbs are being characterized (see presentations by Hayakawa, et al.).