The uptake and distribution of CO throughout the circulatory system during two different methods of CO rebreathing (2 min 'Schmidt' and 40 min 'Burge' methods) were determined in nine healthy volunteers. Specifically, the impact of (i) differences in circulatory mixing time (tmix), (ii) CO diffusion to myoglobin (Mb) and (iii) CO wash-out was assessed on calculated haemoglobin mass (Hbmass). Arterial (a), muscle venous (vm) and capillary samples (c) were obtained simultaneously at 0, 1, 2, 3.5, 5, 7.5, 10, 12.5, 15, 20, 30 and 40 min for determination of carboxyhaemoglobin (HbCO). Carbon monoxide wash-out was measured from expired air following rebreathing. The rate of CO diffusion to Mb was calculated using the change in HbCO after tmix, and the rate of CO wash-out. In both methods, HbCOa and HbCOc followed a near-identical time course, peaking within the first 2 min and decreasing thereafter. The HbCOvm increased slowly and was significantly lower at 1, 2 and 3.5 min in both methods (P < 0.01). The HbCOa peaked significantly higher in the Schmidt method (P= 0.03). Circulatory mixing had occurred by 10 min in most but not all subjects. The rate of CO wash-out was 0.25 ± 0.13 ml min-1 in the Schmidt and 0.25 ± 0.16 ml min-1 in the Burge method. The rate of CO diffusion to Mb was 0.22 ± 0.11 and 0.16 ± 0.13 ml min-1 (P= 0.63) in Schmidt and Burge methods, respectively. Inhalation of a CO bolus during the Schmidt method results in faster HbCOa uptake but does not greatly shorten tmix or influence rates of CO wash-out and flux to Mb. The calculated Hbmass depends substantially on the plateau level of HbCO; therefore, it is paramount to ensure HbCO is mixed completely prior to blood sampling, as well as accounting for potential within-subject alterations of CO exhalation and CO flux to Mb.