Antiserum to dehistonized chromatin from human colon adenocarcinoma cell line HT-29 was raised in rabbits and used for the immunodetection of colon tumor-associated nuclear antigens. Quantitative microcomplement-fixation studies indi cated that the antiserum had reactivity similar to those of chromatins of two human colon adenocarcinoma cell lines (HT29 and LoVo) and with nonneoplastic colon. Immunocytochemical localization showed the chromatin antigens to be evenly dispersed throughout the nucleus. Immunochemical staining of electrophoretically separated chromosomal proteins trans ferred to nitrocellulose sheets revealed several intensely stain ing antigens predominantly in the molecular weight range of 62,000 to 75,000. When the antiserum was immunoabsorbed with nonneoplastic human colon chromatin, two antigens with molecular weights of 67,000 and 92,000 were identified only with the tumor chromatins. Antiserum immunoabsorbed with either LoVo or HT-29 chromatin failed to show any immunoreactivity, suggesting that these tumor-associated proteins, or antigenic sites residing on these proteins, are identical in both colon tumor cell lines.