The purpose of this study was to assess the relationship between progression-free survival (PFS) in patients treated with DFMO + PCV (procarbazine, CCNU, vincristine) chemotherapy for malignant gliomas with tumor cell ornithine decarboxylase (ODC) activity. Formalin-fixed slides were obtained for study patients with anaplastic gliomas (AGs) and glioblastoma treated on protocol DM92-035. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647). Ab-ODC-Alexa 647 intensity in transgenic murine hearts of differing ODC activity was used to calculate ODC activity in tumor cell nucleoplasm. In total, tumor specimens for 31 of 114 (27%) patients treated on the AG strata and 10 patients from the GBM strata were obtained. We found that tumor ODC level heterogeneity increased with increasing tumor malignancy. In a Cox proportional hazards model, PFS was found to be inversely related to median tumor ODC activity, with an unadjusted hazard ratio for median ODC group (>3.3 vs. ≤3.3 nmol/30 min/μg protein) of 5.8 (p < 0.0001); a median PFS of 522 weeks for patients with AGs with median ODC activity ≤ 3.3 and 31 weeks for the 8 AG and 10 glioblastoma patients with ODC activity > 3.3 nmol/30 min/μg protein. Of AG tumors in which ODC activity was evaluated, 26% had ODC levels > 3.3 nmol/30 min/μg protein. This study shows that Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate marker of ODC biochemical activity in AGs and can predict PFS to DFMO-based chemotherapy. © 2007 Wiley-Liss, Inc.