Breath hydrogen testing has a diagnostic potential as a gastrointestinal function test that could be performed in general practice. The purpose of this study was to improve techniques for collection of breath samples and transfer of samples to transport vessels. Breath samples from 10 dogs were collected using both a snug-fitting and a loose-fitting standard anesthetic mask attached to a reservoir bag, and a modified snug-fitting system. CO(2) was used as internal standard and mean CO(2) concentrations were 1.19+/-0.76, 2.17+/-0.66 and 2.68+/-0.92, respectively. Additional samples were saved in transport tubes for 19 days, during which the hydrogen and carbon dioxide concentrations remained stable. A reliable method for transferring the breath samples from the reservoir bag to vacuum transport tubes was also identified. Our results demonstrate less contamination of breath samples with air than previously reported, and a reproducible method to transfer breath samples to transport vessels.