Our headspace gas chromatographic flame ionization detection (HS-GC-FID) method for ethanol determination showed slightly, but consistently, low ethanol concentrations in whole blood (blood) in proficiency testing programs (QC-samples). Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T degrees ) of 70 degrees C and 20 min equilibration time (HS-EqT). Full factorial designs were used to study the variables HS-T degrees (50 degrees -70 degrees C), HS-EqT (15-25 min), ethanol concentration (0.20-1.20 g/kg) and storage at room temperature (0-6 days) with three sample-sets; plasma, hemolyzed blood and non-hemolyzed blood. A decrease in the ethanol concentration in blood was seen as a nearly equivalent increase in the acetaldehyde concentration. This effect was not observed in plasma, indicating chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells. The variables showed different magnitude of effects in hemolyzed and non-hemolyzed blood. A decrease in ethanol concentration was seen even after a few days of storage and also when changing the HS-T degrees from 50 to 70 degrees C. The formation of acetaldehyde was dependent on all the variables and combinations of these (interactions) and HS-T degrees was involved in all the significant interaction effects. Favorable instrumental conditions were found to be HS-T degrees of 50 degrees C and HS-EqT of 15-25 min. The ethanol concentrations obtained for the range 0.04-2.5 g/kg after analyzing authentic forensic blood samples with a HS-T degrees of 50 degrees C were statistically significantly higher than at 70 degrees C (+0.0154 g/kg, p < 0.0001, n = 180). In conclusion, chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells has been shown to contribute to lowered ethanol concentrations in blood samples. Storage conditions before analysis and the headspace equilibration temperature during analysis were important for the determination of blood ethanol concentrations.