We measured single-breath CO transfer factor (TLCO) and alveolar oxygen partial pressure (PAO2) six times at each of three fractions of inspired oxygen (FIO2) (0.17, 0.21, 0.26) in twelve healthy subjects, to determine whether one FIO2 would have the advantage of producing less variable TLCO results than the others. Measured TLCO was adjusted for the increase in carboxyhaemoglobin during the tests. We found no significant differences in intra- or interindividual variance as a function of test FIO2.