BACKGROUND: Nitric oxide (NO) is a compound with both protective and damaging effects on neurons. Quantification of NO metabolites in humans is limited by sample contamination with blood. In vivo cerebral microdialysis may offer an alternative approach as sampling of extracellular fluid (ECF) adjacent to neurons becomes possible. We investigate the prognostic value of brain ECF NO metabolites in patients with traumatic brain injury (TBI). METHODS: A prospective case cohort of 195 ECF samples collected from 11 cases over 4 days following TBI was collected. Nitrate and nitrite concentrations ([NO( x )]) were quantified using a vanadium-based colorimetric assay. RESULTS: Early ECF [NO( x )] (<48 h post TBI) were significantly higher in non-survivors (median 59.2 μmol/l, n = 7) compared to survivors (23.3 μmol/l, n = 4) (P = 0.04). Late (48-96 h) ECF [NO( x )] remained higher in non-survivors (47.9 μmol/l) compared to survivors (23.0 μmol/l) but this was not significant (P = 0.29). Receiver operator characteristic analysis shows an optimized cutoff level for ECF [NO( x )] of 26.5 μmol/l measured <48 h post TBI for predicting non-survival (sensitivity 100%, specificity 75%). CONCLUSION: Early ECF NO( x ) concentrations are of prognostic value after TBI. ECF NO( x ) may be a useful biomarker for treatment trials targeted at nitric oxide metabolism.