It has been claimed that the commercially available Ki-67 monoclonal antibody recognizes a nuclear antigen which is solely expressed in cycling cells. Therefore, at present, Ki-67 is increasingly used as a tool in evaluating growth fractions (GFs) of human tumors. Here we describe specific patterns in the expression and topological distribution of this antigen during the cell cycle in MCF-7 human breast cancer cells. Our results support earlier findings that the antigen belongs to a class of antigens associated with the structural organization of meta- and anaphase chromosomes, and proteins located near the cortical regions of prenucleolar bodies and nucleoli. Using 5'-bromodeoxyuridine-labeling technology, we show that the expression may be undetectably low at the onset of DNA replication. Comparison of Ki-67 fractions (KFs) and GFs as estimated from continuous 5'-bromodeoxyuridine-labeling curves revealed that KF was invariably higher than GF: in exponentially growing cov362.c14 human ovary cancer cells, KF was only 3.5% higher than GF; in MCF-7 cells, 11.3 +/- 4.6%. In MCF-7 cultures either growing under suboptimal conditions or treated with 10(-6) M tamoxifen, the difference was more pronounced. Furthermore, we evaluated the decrease of Ki-67-positive cells in nutritionally deprived and cell cycle-specific, drug-treated cultures. Since the results indicate that nonproliferating cells may retain the antigen for a considerable period of time, KF may not always be a reliable indicator of GF.