What is the central question of this study? Is it possible to modify the CO-rebreathing method to acquire reliable measurements of haemoglobin mass in ventilated patients? What is the main finding and its importance? A 'single breath' of CO with a subsequent 30s breath hold provides almost as exact a measure of haemoglobin mass as the established optimized CO-rebreathing method when applied to healthy subjects. The modified method has now to be checked in ventilated patients before it can be used to quantify the contributions of blood loss and of dilution to the severity of anaemia. Anaemia is defined by the concentration of haemoglobin (Hb). However, this value is dependent upon both the total circulating haemoglobin mass (tHb-mass) and the plasma volume (PV) - neither of which is routinely measured. Carbon monoxide (CO)-rebreathing methods have been successfully used to determine both PV and tHb-mass in various populations. However, these methods are not yet suitable for ventilated patients. This study aimed to modify the CO-rebreathing procedure such that a single inhalation of a CO bolus would enable its use in ventilated patients. Eleven healthy volunteers performed four CO-rebreathing tests in a randomized order, inhaling an identical CO volume. In two tests, CO was rebreathed for 2min (optimized CO rebreathing; oCOR), and in the other two tests, a single inhalation of a CO bolus was conducted with a subsequent breath hold of 15s (Procnew 15s) or 30s (Procnew 30s). Subsequently, the CO volume in the exhaled air was continuously determined for 20min. The amount of CO exhaled after 7 and 20min was respectively 3.1±0.3 and 5.9±1.1ml for oCOR, 8.7±3.6 and 12.0±4.4ml for Procnew 15s and 5.1±2.0 and 8.4±2.6ml for Procnew 30s. tHb-mass was 843±293g determined by oCOR, 821±288g determined by Procnew 15s (difference: P<0.05) and 849±311g determined by Procnew 30s. Bland-Altman plots demonstrated slightly lower tHb-mass values for Procnew 15s compared with oCOR (-21.8±15.3g) and similar values for Procnew 30s. In healthy volunteers, a single inhalation of a CO bolus, preferably followed by a 30s breath hold, can be used to determine tHb-mass. These results must now be validated for ventilated patients.