The influence of the method alveolar air collection on measurements of trace gas concentration has received little attention. We measured the concentrations of H2, CH4, CO, and CO2 in sequential fractions of alveolar air collected with and without breath-holding. Without breath-holding, the concentration of these gases increased appreciably as increasing quantities of alveolar air were expelled. Twenty seconds of breath-holding markedly reduced this nonhomogeneity of alveolar air. Prediction of the excretion rate of trace gases from measurements of their concentration relative to CO2 and literature values for resting CO2 excretion underestimated the true excretion rate. We conclude that breath-holding prior to sample collection enhances the reproducibility of trace gas measurements. When calculating the rate of excretion of trace gases, the use of literature values for resting ventilation or CO2 excretion may result in appreciable underestimations of the true rate.