To examine the role of DNA topoisomerase II (Topo II) in the mitogenic activation of mouse lymphocytes, we applied the Topo II inhibitor VM26 throughout the stimulation period and monitored morphological and functional parameters of lymphocyte activation. Cell viability and the usual increase in cell size were little affected at doses between 0.05 and 0.5 μM. DNA synthesis, however, was already significantly inhibited at 0.05 μM, with RNA synthesis inhibited to a lesser extent. Light microscope autoradiography showed that a smaller proportion of cells entered S phase, with each S phase cell incorporating less [ ³ H]thymidine. In immunofluorescence studies, the nucleolar antigen fibrillarin was reduced, although only minor effects on the snRNP Sm antigen and the internal component labeled by antibody PI1 were observed. At the electron microscope level, nucleoli were remodeled and chromatin became aggregated. At a high dose of VM26 (5 μM), cells showed the expected high levels of apoptosis and strong inhibition in all activation parameters assayed. The results support the hypothesis that the Topo IIβ isoform is involved in the very early phases of lymphocyte activation, with function of the Topo IIα isoform, which is more sensitive to VM26, being required for progression through S phase.