The expression of two novel proliferation-associated markers, mitosin and topoisomerase IIalpha (Topo IIalpha), was evaluated immunohistochemically in consecutive paraffin sections from 60 diffuse astrocytomas (grades 2 to 4) in relation to clinicopathologic parameters, proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1) expression and survival. The percentage of mitosin and Topo IIalpha-positive cells (LI) increased with grade and Ki-67 LI, but could not discriminate between grade 3 on the one hand and grades 2 or 4 on the other hand. In 51% of cases, Ki-67 LI exceeded Topo IIalpha LI, especially within grade 4. Topo IIalpha and mitosin expression was adversely related to overall and disease-free survival in the entire cohort and in grades 2/3. However, only Topo IIalpha LI affected disease-free survival in grade 4 tumors. Multivariate analysis selected only mitosin LI along with the age of the patient, as the independent parameters predicting overall survival, whereas Topo IIalpha emerged as the single independent predictor of disease-free survival. It is concluded that the proliferative potential of astrocytomas, as measured by mitosin and Topo IIalpha immunostaining, conveys useful prognostic information, in addition to that obtained by standard clinicopathologic parameters.