In vitro characterisation results for O2reduction at Pt-based microelectrodes are presented and compared with those for carbon-paste electrodes (CPEs). Cyclic voltammetry indicates a potential of −650 mV vs. SCE is required for cathodic reduction at both electrode types, and calibration experiments at this potential revealed a significantly higher sensitivity for Pt (−0.091 ± 0.006 μAmm−2μM−1vs. −0.048 ± 0.002 μAmm−2μM−1 for CPEs). Since Pt electrodes are readily poisoned through contact with biological samples selected surface coated polymers (polyphenylenediamine (PPD), polymethyl methacrylate (PMMA) and Rhoplex®) were examined in biocompatibility studies performed in protein, lipid and brain tissue solutions. While small and comparable decreases in sensitivity were observed for bare Pt, Pt-Rhoplex and PMMA there was minimal change at the Pt-PPD modified electrode for each 24h treatment, including an extended 3 day exposure to brain tissue. The polymers themselves had no effect on the O2 response characteristics. Further characterisation studies at the Pt-based microelectrodes confirmed interference free signals, no effect of pH and ion changes, and a comparable detection limit (0.08 ± 0.01 μM) and response time (<1 s) to CPEs. Although a significant temperature effect (ca. 3% change in signal for each 1 °C) was observed it is predicted that this will not be important for in vivo brain tissue O2 measurements due to brain temperature homeostasis. These results suggest that amperometric Pt electrodes have the potential to be used reliably as an alternative to CPEs to monitor brain tissue O2 over extended periods in freely-moving animals.