To investigate the growth characteristics of human leukemia cells, the expression of proliferation-associated nuclear antigens was examined in relation to cell cycle phases in marrow blast cells obtained from 37 untreated children with acute leukemia. Ki-67 monoclonal antibody reactive antigen and proliferating cell nuclear antigen (PCNA) were measured by the simultaneous flow cytometric analysis of DNA and nuclear antigens. The percentage of PCNA-positive cells was always higher than that of Ki-67-positive cells in individual patients. The level of PCNA was greatly increased in G1 or early S phase, but was generally stable in S and G2 phases. Accordingly, most of the cells in the proliferative compartments (greater than 2C DNA) showed a high expression of PCNA. In contrast, expression of Ki-67 antigen varied greatly from patient to patient, and differed significantly in different subtypes of the disease. The level of Ki-67 antigen increased with the cell cycle progression, showing maximum expression in late S and G2 phases. However, in most of the patients, a distinct population of Ki-67-negative cells was found not only in G1 phase, but also in the proliferative compartments. These results appear to reflect differences in the proliferative activity of bone marrow blast cells in childhood acute leukemia.