The objective of this study was to generate data that allow for estimation of the validity of oxygen saturation (SO2) values in superficial cortical capillaries as calculated by a microreflectometric system (EMPHO II). Capillary SO2 and tissue oxygen pressure (PtO2) were measured simultaneously in the cortex of n = 13 Wistar rats under normocapnic (PaCO2 = 36 mmHg) arterial normoxia (PaO2 = 92 mmHg), moderate (paO2 = 53 mmHg) and severe hypoxic hypoxia (PaO2 = 31 mmHg) with microreflectometry and multiwire surface electrodes. Values were pooled according to arterial oxygenation levels, displayed as frequency histograms and compared via ANOVA (p < 0.05). In a Hill-plot (log PtO2 versus log SO2/(100 - SO2)) an in vivo tissue oxygen dissociation curve was obtained and a linear regression/correlation analysis performed. Mean +/- SD values of SO2 respectively PtO2 decreased from 45.6% +/- 14.6% resp. 26.8 +/- 8.2 mmHg during arterial normoxia to 32.6% +/- 10.2% resp. 20.2 +/- 6.6 mmHg during moderate and to 12.3% +/- 11.1% resp. 8.7 +/- 5.0 mmHg during severe hypoxic hypoxia. Linear regression analysis in the Hill-plot of values between 1% and 65% SO2 and 0.1 and 41 mmHg PtO2 revealed an excellent correlation (r2 = 0.88) with an increase of scatter below 10% SO2 or 1.5 mmHg PtO2. We conclude that SO2 values calculated by the algorithm of the applied microreflectometric system reflect very accurately cortical oxygen supply over a very wide range of oxygenation levels when compared to a gold standard reference. Only at extremely low levels (e.g. below 10% SO2) did we find possible inaccuracies with regard to truly absolute saturation values.