The present study reports the identification and partial characterization of a novel Mr 40,000 nucleolar antigen (P40) by monoclonal antibodies. Monoclonal antibodies to this protein were obtained when a nucleolar protein extract separated from the immunodominant protein C23 was used to immunize BALB/c mice; 12 hybridoma clones produced antibodies to this protein. P40 was not detected in normal human kidney, liver, and leukocytes but was readily demonstrable in a variety of human malignant tissues. This newly identified P40 antigen differs in its specific nucleolar localization from cyclin (proliferating cell nuclear antigen), a Mr 35,000 antigen which is largely in the nucleoplasm. In addition, cyclin appears in the nucleolus in S-phase; P40 appears in the nucleolus 6 h after refeeding serum-starved HeLa cells.