Oxidative stress refers to an imbalance between the production of oxidising free radicals and the antioxidant defenses of the cell, and is associated with many pathogenic processes. Oxidative damage to cellular lipids results in the evolution of pentane and ethane gas, and detection of these hydrocarbons in the exhaled breath can be used to monitor in vivo oxidative stress. The aim of this study was to validate a gas chromatography (GC) method for measurement of breath pentane in the horse. The GC-system developed showed good specificity for discrimination of pentane from other breath hydrocarbons, and was sensitive to 0.5 ppb pentane. Pentane was detected in the exhaled breath of five horses investigated on two occasions. The results of this preliminary study demonstrate that breath pentane can be measured in the horse, and further work is now justified to investigate the feasibility of applying this method for monitoring in vivo oxidative stress in the horse.