The isolation of monoclonal antibiotics to chromatin-associated protein antigens and their use in the characterization of such proteins by indirect immunofluorescence are described. Hybridomas were derived by fusion of the mouse myeloma Ag8653 with spleen cells from mice immunized with chromatin from human liver, rat liver or a human lymphoblastoid cell line. Hybrids were screened by solid-phase radioimmunoassay. The proportion of positive hybrids varied with the immunizing chromatin as follows: human liver 55/83, human lymphoblast 8/183 and rat liver 2/82. Fifteen antibodies derived from these fusions (7, 7 and 1, respectively) were subjected to further analysis. Most of these (11/13) were IgM and recognized both human and rat chromatin (12/15). Most of the target antigens were protease sensitive (8/13) and nuclease resistant. The binding of 5 antibodies to lymphoblast chromatin was more than doubled by preincubation with DNase I. The subcellular location of target antigens was examined by indirect immunofluoresence. Seven antibodies stained at least 1 of several cultured cell lines tested. Three gave staining patterns consistent with the in vivo association of the target antigen with chromatin recognizing the interphase nucleus and metaphase chromosomes, the nuclear periphery and the mitotic spindle and other microtubule-containing structures. The remaining 4 all recognized antigens associated with the intermediate filament network.