5136 Background: Tumor hypoxia may confer radioresistance in prostate cancer. The purpose of this study was to correlate tumor oxygenation status with long term biochemical outcome following prostate bracytherapy. Methods: Custom made Eppendorf PO2 microelectrodes were used to obtain PO2 measurements from the prostate (P), focused on + biopsy locations, and normal muscle tissue (M), as a control, in the operating room using a sterile technique. A total of 11,516 measurements were obtained in 57 patients with localized prostate cancer immediately prior to prostate brachytherapy. The Eppendorf histograms provided the median PO2, mean PO2, and % < 5 mm Hg or < 10 mm Hg. All patients received brachytherapy implants (48 low dose rate and 9 high dose rate) and five patients had received prior hormonal therapy. Biochemical failure (BF) was defined using both the newer Phoenix (PSA nadir + 2 ng/mL) the prior ASTRO (three consecutive rises) definitions (dfn). A Cox proportional hazards regression model evaluated the influence of hypoxia on BF with P/M ratio as a continuous variable and dichotomized at 0.05, 0.10, and 0.20. Results: The median follow-up was 8 years (range: 0.8- 9.4 years). Twelve patients developed BF via ASTRO dfn and 8 via Phoenix dfn. On stepwise multivariate analysis, P/M ratio < 0.10 was a significant predictor of BF (ASTRO, p = 0.03; Phoenix p = 0.02) in a model including initial PSA, Gleason score, T-stage, perineural invasion, age, hemoglobin and use of hormonal therapy. Kaplan-Meier freedom from BF for P/M ratio < 0.10 vs. ≥ 0.10 at 8 years was 46% vs 78% (p = 0.03) for the ASTRO dfn and 66% vs 83% (p = 0.02) for the Phoenix dfn. Only one patient (in the P/M ratio < 0.10) manifested distant failure. Conclusions: Hypoxia in prostate cancer (low P/M PO2 ratio) significantly predicts for poor long term biochemical outcome on multivariate analysis, suggesting that novel hypoxic strategies should be investigated. No significant financial relationships to disclose.