The nuclear isoform of deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, OMIM *601266, EC 3.6.1.23) is immunohistochemically detectable in all proliferating tissues and may thus be a useful adjunct for the grading of tumors analogous to Ki-67 labeling. A hundred and twenty-seven human intracranial tumors, including 56 astrocytomas, 12 oligodendrogliomas, 8 oligoastrocytomas, 34 meningiomas, 7 ependymomas, and 10 metastatic carcinomas, were stained using the monoclonal rat anti-human dUTPase antibody (clone 3E6) with formalin-fixed and paraffin-embedded tissue. The labeling indices were compared with those obtained with the proliferation marker Ki-67 on parallel tissue sections. All tumors contained dUTPase-positive nuclei, whereas the percentage of positive tumor cells generally increased with grade of malignancy. Meningiomas of higher grades, i.e., World Health Organization (WHO) grades II and III, contained additional cells with cytoplasmic reactivity. There were usually fewer dUTPase- than Ki-67-positive nuclei detectable. Unlike Ki-67, dUTPase was not detectable in mitotic figures. Labeling indices for dUTPase, but not for Ki-67, showed significant differences between all 3 WHO grades of diffuse astrocytomas. In summary, dUTPase staining provides a useful measure of cell proliferation distinct from that offered by Ki-67 labeling. It proved particularly useful for the evaluation of diffuse astrocytomas.