In contrast to the standard single-breath transfer factor for carbon monoxide (TLCO), there are no specific guidelines or recommendations for the measurement of its components, the pulmonary capillary blood volume (VC) and membrane component (DM), by the Roughton and Forster method. Ten randomly selected heart transplant patients (three life-long non-smokers, seven ex-smokers > 1 yr, age range 24-55 years) were assessed on two occasions using either the standard or high-oxygen mixture as the first inspired gas in random order. Ten normal subjects (all non-smokers, age range 23-54 years) were assessed on two occasions using either a long protocol (30 min waiting time between repeat measurements in an individual set) or a short protocol (5 min waiting time). Two technically acceptable results of TLCO were used to derive a mean value for DM and VC for each set of measurements (Transflow, P. K. Morgan, Kent, U.K.). The different sequences of gas mixtures produced no significant differences between the values obtained in ten heart transplant patients for mean TLCO (mmol min-1 kPa-1) (standard first 5.13 +/- 1.15, high-oxygen first 5.14 +/- 1.12; limits of agreement -0.57 to 0.59 for DM or for VC. The long or short protocol produced no significant differences between the means of TLCO (mmol min-1 kPa-1) (long 8.0 +/- 1.9, short 8.0 +/- 1.9; limits of agreement -0.5 to 0.5), DM or VC. This allows the development of a standard test protocol of short duration (about 40 min) making it practical for clinical use without compromising the precision or reproducibility of the results obtained.